Treatment of post-bariatric hypoglycemia with exendin (9-39)

ABSTRACT

Compositions and methods for treating hyperinsulinemic hypoglycemia, such as hyperinsulinemic hypoglycemia after bariatric surgery, are provided. In some embodiments, an effective amount of the glucagon-like peptide-1 receptor antagonist exendin(9-39) is subcutaneously administered twice per day.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/813,535, filed Mar. 9, 2020, which is a continuation of U.S.application Ser. No. 15/576,647, filed Nov. 22, 2017, which is theNational Stage of International Application No. PCT/US2016/033837, filedMay 23, 2016, which claims priority to U.S. Provisional Application No.62/165,743, filed May 22, 2015, U.S. Provisional Application No.62/254,175, filed Nov. 11, 2015, and U.S. Provisional Application No.62/329,850, filed Apr. 29, 2016, the contents of each of which areincorporated by reference in their entireties for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under contract TR001085awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention provides methods and compositions for thetreatment of hypoglycemia, particularly post-bariatric hyperinsulinemia,and more generally hyperinsulinemic hypoglycemia of any origin, and theprevention of associated acute symptoms and chronic outcomes in which aglucagon-like peptide-1 receptor antagonist (GLP1A), exendin(9-39), issubcutaneously administered in a therapeutically effective dose. Theinvention therefore relates to the fields of biology, chemistry,medicinal chemistry, medicine, molecular biology, and pharmacology.

BACKGROUND OF THE INVENTION

Roux-en-Y gastric bypass (RYGB), widely performed for medicallycomplicated obesity, cures type 2 diabetes in 85% of cases. Thephysiologic mechanisms mediating diabetes resolution is controversial,but the reduction in glucose excursions prior to weight loss has led topostulates that the incretin hormone, glucagon-like peptide-1 (GLP-1),may play an important role. GLP-1 stimulates the secretion of insulin bypancreatic beta cells and is responsible for the “incretin” effect:incretin hormones enhance the glucose-dependent secretion of insulin,such that pancreatic beta cells will secrete more insulin after an oralglucose load than after an isoglycemic IV glucose load. Enhancedsecretion of GLP-1 after RYGB, and a resultant elevation in insulinsecretion, may play a primary role in the resolution of diabetes afterRYGB. Indeed GLP-1 analogs have been developed to treat diabetes.

However, as the use of bariatric surgical procedures continues toincrease worldwide, a severe complication—hyperinsulinemichypoglycemia—is increasingly reported. Present in 1-6% of RYGB patients,this disorder leads to severe symptomatic hypoglycemia that plaguespatients often multiple times daily, with glucose concentrations lowenough (20-40 mg/dL) to cause seizures, altered mental status, loss ofconsciousness, cognitive dysfunction, disability, and death. Quality oflife is severely diminished, and many patients cannot care forthemselves or others, work, drive, or be left alone. There is noeffective treatment and severe cases have been managed with near-totalto total pancreatectomy, which results in insulin-dependent diabetes andis associated with a 6% surgical mortality risk.

Given the severity of this chronic disorder with unmet clinical need, aneffective therapeutic treatment is urgently needed. It would thus beuseful to provide a method for the treatment of hyperinsulinemichypoglycemia post bariatric surgery and prevention of its acute symptomsand chronic outcomes, and a pharmaceutical composition for suchtherapeutic.

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical compositions and methodsinvolving twice-per-day subcutaneous delivery of exendin(9-39) in dosestherapeutically effective for treating or preventing hyperinsulinemichypoglycemia in a patient who has previously had bariatric surgery, suchas Roux-en-Y gastric bypass surgery. The twice-per-day administration isgenerally a first administration in the morning and a secondadministration in the evening. In a particular embodiment thetwice-per-day subcutaneous delivery involves administering a compositioncomprising a therapeutically effective amount of exendin(9-39), whereinthe therapeutically effective amount is in the range of 10 mg-30 mgexendin(9-39). In some embodiments the therapeutically effective amountof exendin(9-39) administered twice-per-day is in the range of 10-20 mg(e.g., 10 mg, 15 mg, or 20 mg), in the range of 10 mg-15 mg (e.g., 10 mgor 15 mg), or in the range of 15 mg-30 mg (e.g., 15 mg, 20 mg, 25 mg, or30 mg). The exendin(9-39)-containing composition may be a solution orsuspension that comprises exendin(9-39) at a concentration in the rangeof 4-20 mg/ml. In some embodiments the concentration is in the range of10-20 mg/ml. In some embodiments the concentration is in the range of8-16 mg/ml. In some embodiments the concentration is in the range of13-16 mg/ml.

In some approaches the composition, or injectate, administered in themorning administration and the composition, or injectate, administeredin the evening administration are the same, i.e., they contain the sameamount (dose) of exendin(9-39) at the same concentration. In anotherapproach subcutaneous administration twice-per-day comprisesadministering a morning injectate and an evening injectate, where theamount and/or concentration of exendin(9-39) in the evening injectate isgreater than the amount of exendin(9-39) in the morning injectate. Inone approach the amount of exendin(9-39) in the evening injectate isgreater than the amount of exendin(9-39) in the morning injectate. Inone approach the concentration of exendin(9-39) in the evening injectateis higher than the concentration of exendin(9-39) in the morninginjectate.

In some cases, the amount of exendin(9-39) in the evening injectate is 5mg-10 mg greater than the amount in the morning injectate. In some suchcases the amount of exendin(9-39) in the morning injectate is 10 mg andthe amount in the evening injectate is 15 mg. In some such cases theamount of exendin(9-39) in the morning injectate is 10 mg and the amountin the evening injectate is 20 mg. In some such cases the amount ofexendin(9-39) in the morning injectate is 15 mg and the amount in theevening injectate is 20 mg. In some such cases the amount ofexendin(9-39) in the morning injectate is 15 mg and the amount in theevening injectate is 25 mg. In some such cases the amount ofexendin(9-39) in the morning injectate is 20 mg and the amount in theevening injectate is 25 mg. In some such cases the amount ofexendin(9-39) in the morning injectate is 20 mg and the amount in theevening injectate is 30 mg

In some cases, the concentration of exendin(9-39) in the morninginjectate is not the same as the concentration of exendin(9-39) in theevening injectate. For example, in one approach the concentration ofexendin(9-39) in morning injectate is 15 mg/ml and the concentration ofexendin(9-39) in the evening injectate is 20 mg/ml.

In some cases the exendin(9-39) amount and concentration of the morningand evening injectates are selected such that the exendin(9-39) Tmaxafter the evening administration is longer than the Tmax after themorning administration.

In some cases the amount and concentration of the morning and eveninginjectates are selected such that the administration results in anexendin(9-39) Cmax of at least 100 ng/ml (e.g., as measured by liquidchromatograph-mass spectrometry).

In some approaches administration comprises a first daily administrationis in the morning and a second daily administration in the evening,where the second daily administration is at least 8 hours after thefirst daily administration. In some such cases the morningadministration is administered before the morning meal (e.g.,breakfast). In some such cases the morning administration isadministered at least 1 hour before the morning meal, optionally 60 to90 minutes before the morning meal. In some cases the morningadministration is in the period from one hour before the morning meal toone hour after the morning meal. In some cases the second dailyadministration is administered 60 to 90 minutes before the evening meal.In some cases the second daily administration is administered after theevening meal and before bedtime (for example, within 2 hours ofbedtime). In some cases, the evening administration is from 9 to 15hours after the morning administration.

In some embodiments, a dose of exendin(9-39) is administered with regardto the timing of a meal. For example, in some embodiments, the dose ofexendin(9-39) is administered with a meal, or before a particular meal,including, for example a certain time, e.g., 15 minutes to two hours,e.g. one hour, before a meal, or a certain time after a meal.

In some embodiments, a dose of exendin(9-39) is administered withoutregard to the timing of a meal.

In one embodiment of the invention, the patient self-administers (or iswearing a device programmed to administer) the exendin(9-39). In someembodiments, the first dose may be administered in the evening, suchthat it provides protection during breakfast the following day, withsubsequent doses following the next morning and the next evening abouttwelve hours later.

In some embodiments, exendin(9-39) is formulated and administered in aninjectable pen device or via a vial/syringe combination that may bepre-programmed or marked to deliver a fixed dosage amount (andoptionally two different fixed dosage amounts corresponding to morningand evening administrations) ranging from 10-30 mg exendin(9-39).

The pharmaceutical compositions of the invention have use for treatmentand prevention of hyperinsulinemic hypoglycemia and its associatedsymptoms and outcomes in patients with hyperinsulinemic hypoglycemiapost bariatric surgery and post gastrointestinal surgery. In these andother embodiments, the invention provides for the prevention andtreatment of associated acute and chronic symptoms and outcomes insusceptible patients. Treatment in accordance with the invention ofpatients in need of therapy will improve patient quality of life both inthe short- and long-term, will reduce overall patient morbidity, and mayprevent premature death and/or extend life-expectancy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B. Average plasma glucose (A) and insulin (B) responses to a 75gram oral glucose tolerance test (OGTT) in subjects withhyperinsulinemic hypoglycemia during a randomized blinded cross-overstudy in which a primed continuous intravenous (IV) infusion ofexendin(9-39) (at 500 pmol/kg/min over 180 minutes) or placebo (normalsaline) was administered, as described in Example 1. Solid line: placeboinfusion; dashed line: exendin(9-39) infusion.

FIG. 2A-C. Average plasma GLP-1 (A), GIP (B), and glucagon (C) responseto a 75 gram OGTT in subjects with hyperinsulinemic hypoglycemiareceiving primed continuous IV infusion of exendin(9-39) of 500pmol/kg/min over 180 minutes versus placebo (normal saline) infusion, asdescribed in Example 1. Solid line: placebo infusion; dashed line:exendin(9-39) infusion.

FIG. 3 . Individual and average symptomatic responses to a 75 gram OGTTin 8 patients with hyperinsulinemic hypoglycemia receiving a primedcontinuous IV infusion of exendin(9-39) versus placebo, as described inExample 1. Overall Symptom Score, Glucose Rise, and Glucose Fall Scoresare presented. Continuous IV infusion of exendin(9-39) at 500pmol/kg/min over 180 minutes substantially improved symptoms ofhypoglycemia, as demonstrated by the reduced Overall Symptom and GlucoseFall scores.

FIG. 4A-D. Plasma glucose responses to IV bolus doses of 7,500 pmol/kgof exendin(9-39) administered to a subject with hyperinsulinemichypoglycemia at different time points relative to the timing ofadministration of glucola in an OGTT. Specifically, the exendin(9-39) IVbolus was administered at: (A) T=0 minutes, (B) T=20 minutes, or (C)T=50 minutes. For (A)-(C), glucose levels measured at specifictimepoints as described in Example 2 are shown by a solid line. For(A)-(D), the projected exendin(9-39) PK curve after administration ofthe IV bolus of 7,500 pmol/kg is shown by a dotted line. Dosing at 0minutes (A) or 20 minutes (B) did not prevent hypoglycemia, whereasdosing at 50 minutes (C) did prevent hypoglycemia. (D) The GLP-1 peakfor the subject in (C) occurred at 60 minutes, suggesting that timingthe administration of the IV bolus to the GLP-1 plasma peak wasnecessary to prevent hypoglycemia.

FIG. 5A-B. (A) Average plasma exendin(9-39) concentrations for 8 humansubjects administered a continuous exendin(9-39) IV infusion at a rateof 500 pmol/kg/min over 180 minutes are plotted (black line). Theprojected exendin(9-39) pharmacokinetic response to a single IV bolus of7,500 pmol/kg exendin(9-39) administered at T-30 (blue line) wasextrapolated based on the known half-life of intravenously administeredexendin(9-39). (B) A single IV bolus of 7,500 pmol/kg exendin(9-39) or asingle subcutaneous injection of 7,500 pmol/kg exendin(9-39) wasadministered to a subject. Plasma exendin(9-39) concentrations weremeasured by liquid chromatography-mass spectrometry (LCMS). The Cmaxthat was observed in subcutaneous administration of exendin(9-39) wassignificantly lower than the Cmax observed in intravenous administrationof exendin(9-39).

FIG. 6 . Average plasma glucose levels during a 75 gram OGTT forsubjects administered a subcutaneous dose of exendin(9-39) as comparedto baseline. Four subjects received one subcutaneous injection of 35,700pmol/kg, 75,000 pmol/kg, or 112,500 pmol/kg (approximately 10, 20, or 30mg, respectively, based on an 80 kg patient) in a volume of 0.7 mlnormal saline, and four subjects received two or more 0.7 ml injectionsof 35,700 pmol/kg, 75,000 pmol/kg, or 112,500 pmol/kg in order tomaintain an injectate concentration of 15 mg/ml or less. The averageplasma glucose nadir for all 8 subjects during subcutaneous injection ofexendin(9-39) was 78 mg/dL vs. <50 mg/dL during a baseline oral glucosetolerance test, demonstrating that subcutaneous injection of a singledose about 10-30 mg exendin(9-39) was able to effectively reversehyperinsulinemic hypoglycemia.

FIG. 7A-B. Plasma exendin(9-39) concentrations following subcutaneousinjection of exendin(9-39). (A) Three subjects received a singlesubcutaneous injection of approximately 10, 20, or 30 mg ofexendin(9-39) in a volume of 0.7 ml (5×, 10×, or 15× doses,respectively). (B) Five subjects received doses of approximately 2, 10,20, or 30 mg (1×, 5×, 10, or 15×, respectively), with each doseadministered at a concentration of 15 mg/ml or less; higher doses wereadministered via more than one injection so as to maintain a relativelydilute concentration.

FIG. 8 . Percent increase in plasma glucose nadir concentrations werecalculated for the subcutaneously administered doses of exendin(9-39)relative to baseline. A correlation was observed between higher percentincreases in plasma glucose nadir concentrations and increasing peakplasma exendin(9-39) concentrations (Cmax).

FIG. 9 . Study design for 3-day Multi-Ascending Dose Trial to assess thesafety, tolerability, efficacy, and pharmacokinetic profile of BIDexendin(9-39) administered subcutaneously over 3 days to patients withpost-bariatric hyperinsulinemic hypoglycemia.

FIG. 10 . Exendin(9-39) plasma concentrations on Day 3 after 5 doses asdescribed in Example 4.

BRIEF DESCRIPTION OF THE TABLES

Table 1: Metabolic responses to a 75 gram oral glucose tolerance test(OGTT) during primed continuous IV infusion of exendin(9-39) in eightpost-RYGB patients with hyperinsulinemic hypoglycemia (HH). Metabolicresponses of eight BMI, age, and sex matched non-surgical controls arepresented for comparison. AUC values were calculated by the trapezoidalrule utilizing the last value carried forward to account for prematurelydiscontinued OGTTs in cases of hypoglycemia, which occurred solelyduring placebo infusion.

Table 2: Mean plasma GLP-1, GIP and glucagon response to a 75 gram oralglucose tolerance test (OGTT) in eight patients with hyperinsulinemichypoglycemia (HH) during a primed continuous IV infusion ofexendin(9-39) of 500 pmol/kg/min over 180 minutes vs. during placebo(normal saline) infusion.

Table 3: Subject metabolic and symptomatic response to a singlesubcutaneous (SC) injection of 10-30 mg of exendin(9-39), denoted as SCEx(9-39), continuous IV infusion of exendin(9-39 (IV Ex(9-39)), orplacebo during a 75 gram OGTT. This table demonstrates that clinicalefficacy during this SAD subcutaneous injection study was comparable tothat achieved during continuous IV infusion of exendin(9-39), asmeasured by the plasma glucose nadir, AUC glucose, and the Symptom FallScore.

Table 4: PK/PD response to increasing doses/increasing concentrations.As described in Example 3, subjects 2-5 each received a subcutaneousinjection of exendin(9-39) in doses ranging from 37,500-112,500 pmol/kg(approximately 10-30 mg) each in a volume of 0.7 ml, resulting in doseconcentrations of approximately 15-40 mg/ml. Shown here are subjectPK/PD responses to each dose. Injectate concentrations of approximately15 mg/ml resulted in the greatest pharmacodynamic response, as definedby nadir postprandial glucose and AUC glucose, and greatestpharmacokinetic response, as defined by Cmax and DN Cmax. Thus arelatively dilute dose may be preferred for BID dosing, and a moreconcentrated formulation may be preferred for less frequent dosing or amore sustained exposure. The 75,000 pmol/kg dose (17 mg) with aconcentration of about 24 mg/ml resulted in a favorable sustained/slowrelease pharmacokinetic response, with a half-life of 9.14 hours, and aCmax that was 70 or more ng/ml. Thus a relatively concentrated dose maybe used advantageously for qD or BID dosing not tied to meals.

Table 5: PK/PD response to increasing dose with constant injectateconcentration. As described in Example 3, four subjects receivedsubcutaneous injections of 37,500-112,500 pmol/kg exendin(9-39) inequivalent concentrations (approximately 13-16 mg/ml), as thisconcentration was found to result in a favorable immediate releaseformulation of the invention. Results shown demonstrate an increasinglyfavorable PK response with increasing dose, as defined by Cmax andT_(1/2).

Table 6: PK/PD response in four subjects dosed with varying doses ofsubcutaneously administered BID exendin(9-39) in a 3-day clinical trialas described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION 1. Introduction

Post-bariatric hyperinsulinemic hypoglycemia is a disorder that ischaracterized by low blood sugar and elevated insulin levels 1-3 hoursafter meals. The disorder manifests as neuroglycopenic symptoms (such asconfusion, loss of focus, fatigue, ataxia, paralysis, seizures, or lossof consciousness), vasomotor symptoms (such as sweating and shakiness),and/or adrengeric symptoms (such as heart palpitations). Although thepathogenesis of post-bariatric hyperinsulinemic hypoglycemia is notentirely understood, several mechanisms have been proposed, includingincreased secretion of insulinotropic incretin gut hormones from thehindgut, expansion in (3-cell mass, enhanced (3-cell sensitivity,increased insulin sensitivity, decreased insulin clearance, reducedability to mount a counterregulatory glucagon response, and absence of aprodiabetogenic/decretin foregut factor.

As described in Example 1, in patients having hyperinsulinemichypoglycemia after gastric bypass surgery, blockade of the Glucagon-likePeptide-1 (GLP-1) receptor by administration by primed continuousintravenous (IV) infusion of exendin(9-39) effectively reversedhyperinsulinemic hypoglycemia and associated symptoms. In this trialpatients received a total dose about 24-39 mg exendin(9-39), with thedrug quantity varying with patient weight. (Also see Salehi et al.,2014, “Blockade of Glucagon-like Peptide 1 Receptor CorrectsPostprandial Hypoglycemia After Gastric Bypass,” Gastroenterology146:669-680.) However, because post-bariatric hyperinsulinemichypoglycemia is a chronic, lifelong disorder, the administration of acontinuous IV infusion of exendin(9-39) is not a practical treatmentoption.

Exendin(9-39) administered intravenously is characterized by a shortplasma half-life of 33 minutes (see, Gardiner et al., JPET 316:852-859(2006); see also, Edwards et al., Diabetes 48:86-93 (1999)). Asdescribed in Example 2, below, exendin(9-39) administered as a single IVbolus of 7,500 pmol/kg prevented hypoglycemia in patients only if thebolus was timed to closely coincide with peak GLP-1 plasmaconcentrations. The pharmacokinetic properties exhibited from theadministration of a single intravenous dose of exendin(9-39) are suchthat a person of ordinary skill in the art would not have expectedsubcutaneous administration of clinically appropriate doses ofexendin(9-39) to be therapeutically effective in the treatment ofpostprandial hyperinsulinemic hypoglycemia.

As detailed herein, it has been surprisingly shown that administrationof exendin(9-39) by twice-per-day (BID) subcutaneous injection (SC) caneffectively prevent hypoglycemia in patients having post-bariatrichyperinsulinemic hypoglycemia, that such prevention results at a dose of30 mg or lower (e.g., in the range 7.5 mg-20 mg, e.g., 10 mg-15 mg),that such prevention can be achieved using a convenient administrationschedule not necessarily tied to meal times, and that thepharmacokinetics of exendin(9-39) SC administration may be tuned basedon the concentration of exendin(9-39) in the injectate, as well as bydose, to achieve an efficacious treatment. See Example 3 and Example 4.Thus, in one aspect, the present invention relates to pharmaceuticalcompositions and methods for subcutaneously administering exendin(9-39)at a BID dose in the range of about 7.5 mg-20 mg for the treatment andprevention of hyperinsulinemic hypoglycemia.

It will be recognized by physicians and pharmacologists that the presentinvention represents a significant advance in the field of surgicalintervention for weight loss and/or metabolic control. This isespecially important, because those post-bariatric patients currentlysuffering hypoglycemic excursions have no effective therapy and aresometimes critically ill. The intractable nature of the problem has beenhighlighted by those patients with disease so debilitating they reversedthe surgery, or underwent other highly morbid procedures, such aspartial pancreatectomy, only to learn the condition persists. Thepresent invention provides a therapeutic intervention that can largelyprotect them should they suffer from post-bariatric hyperinsulinemia.

2. Definitions

The terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention pertains. In some cases, terms with commonlyunderstood meanings are defined herein for clarity and/or for readyreference, and the inclusion of such definitions herein should not beconstrued as representing a substantial difference over the definitionof the term as generally understood in the art.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Alltechnical and patent publications cited herein are incorporated hereinby reference in their entirety.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1 or 1.0, as appropriate. It is tobe understood, although not always explicitly stated, that all numericaldesignations are preceded by the term “about.” References to rangesinclude the end-points unless indicated otherwise. For example,administration of a dose of exendin(9-39) in the range 7.5 mg-15 mgincludes administration of 7.5 mg or 15 mg.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a compound” includes a plurality of compounds.

The term “comprising” is intended to mean that the compositions andmethods include the recited elements, but not excluding others.“Consisting essentially of” shall mean excluding other elements thatwould materially affect the basic and novel characteristics of theclaimed invention. “Consisting of” shall mean excluding any element,step, or ingredient not specified in the claim. Embodiments defined byeach of these transition terms are within the scope of this invention.

“Exendin(9-39)” or “Ex(9-39)” or “Ex9” refers to a 31 amino acid peptidewith an empirical formula of C₁₄₉H₂₃₄N₄₀O₄₇S and a molecular weight of3369.8 Daltons. The amino acid sequence for exendin(9-39) is shown asfollows:H-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂.Exendin(9-39) comprises residues 9-39 of the GLP-1 receptor agonistexendin-4 and is a GLP-1 receptor antagonist. See, Montrose-Rafizadeh etal., Journal of Biological Chemistry, 272:21201-21206 (1997). As usedherein, the term “exendin(9-39)” encompasses pharmaceutically acceptablesalts of exendin(9-39), including but not limited to sulfate,hydrochloride, phosophate, sulfamate, acetate, citrate, lactate,tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, cyclohexylsulfamate and quinate salts. In someembodiments, exendin(9-39) is in the form of exendin(9-39) acetate orexendin(9-39) trifluoroacetate. Where not otherwise specified herein,exendin(9-39) acetate is used (obtained from Bachem (Clinalfa,Laufelfingen, Switzerland)).

The abbreviation “GLP1A” refers to a GLP1 receptor antagonist (sometimesreferred to as a “GLP1 antagonist”).

The terms “administer,” “administering,” and “administration,” as usedherein, refer to introducing a compound (e.g., exendin(9-39)) orcomposition into a human subject. As used herein, the terms encompassboth direct administration (e.g., administration to a subject by amedical professional or other caregiver, or by self-administration, orby programming an automatic device to deliver exendin(9-39) on a BIDschedule) and indirect administration (e.g., the act of prescribing acompound or composition to a subject).

The terms “treatment,” “treating,” and “treat,” as used herein inreference to administering exendin(9-39) to treat hyperinsulinemichypoglycemia, covers any treatment of a disease in a human subject, andincludes: (a) reducing the risk, frequency or severity of hypoglycemicepisodes in patients with a history of hyperinsulinemic hypoglycemia,(b) reducing the risk of occurrence of hypoglycemia in a subjectdetermined to be predisposed to the disease, such as a person who hasreceived post-bariatric surgery, but not yet diagnosed as having thedisease, (c) impeding the development of the disease; and/or (d)relieving the disease, i.e., causing regression of the disease and/orrelieving one or more disease symptoms.

As used herein, the term “injectate” refers the exendin(9-39)-containingcomposition subcutaneously delivered to a patient at a morning orevening administration. A morning or evening injectate is typicallyadministered as a single injection (e.g., injection of a 0.7 ml volume).However an injectate can be delivered using more than one (e.g., two)injections, as may be done when the injectate volume is greater thancomfortably tolerated as a single injection.

As used herein, “/kg” (e.g., 7,500 pmol/kg”) means “per kilogram patientbody weight.”

3. Methods and Compositions for the Treatment of HyperinsulinemicHypoglycemia

In one aspect, the present invention provides methods and compositionsfor the treatment of hyperinsulinemic hypoglycemia by subcutaneous (SC)administration of a therapeutically effective dose of exendin(9-39).

3.1 Patient Population

In some embodiments, a patient to be treated according to the methodsdescribed herein is a patient having hyperinsulinemic hypoglycemia (HH).In certain embodiments, the patient having hyperinsulinemic hypoglycemiahas previously had bariatric surgery (e.g., Roux-en-Y Gastric Bypass)and/or a related metabolic procedure. In certain embodiments, thepatient has previously had bariatric surgery (e.g., Roux-en-Y GastricBypass) and/or a related metabolic procedure and is at risk fordeveloping hyperinsulinemic hypoglycemia.

Patients with hyperinsulinemic hypoglycemia may be identified by anysuitable method. In some embodiments, hyperinsulinemic hypoglycemia isdiagnosed by the presence of Whipple's triad, which has the followingcriteria: (1) the occurrence of hypoglycemic symptoms; (2) documentedlow plasma glucose level at the type of the symptoms; and (3) resolutionof the symptoms after plasma glucose is raised. In some embodiments,hyperinsulinemic hypoglycemia is defined by the occurrence of capillaryglucose≤50 mg/dL at least once per month by patient report or medicalrecord. In some embodiments, hyperinsulinemic hypoglycemia is defined bya plasma glucose concentration of ≤55 mg/dL during an oral glucosetolerance test or meal tolerance test in association withinappropriately elevated plasma insulin (≥3 uU/mL) or c-peptide (>0.3mg/dL) when glucose was ≤55 mg/dL. In some embodiments, hyperinsulinemichypoglycemia is defined by a plasma glucose concentration of ≤60 mg/dLduring an oral glucose tolerance test or meal tolerance test inassociation with inappropriately elevated plasma insulin (≥3 uU/mL) orc-peptide (>0.3 mg/dL) when glucose was ≤60 mg/dL.

“Hyperinsulinemic hypoglycemia,” as used herein, encompasses theconditions dumping syndrome, nesideoblastosis, noninsulinomapancreatogenous hypoglycemia syndrome (NIPHS), and/or post-prandialreactive hypoglycemia. Hyperinsulinemic hypoglycemia may result from agastric or bariatric procedure, such as a Roux-en-Y gastric bypass(RYGB), or may have a congenital, acquired, or induced origin.

In one embodiment, the patient treated has previously had a bariatricprocedure and/or related metabolic procedure, such as a Roux-en-YGastric Bypass procedure. Bariatric and/or related metabolic proceduresinclude, but are not limited to, Roux-en-Y Gastric Bypass, VerticalSleeve Gastrectomy, placement of an endosleeve device, such as theEndoBarrier Gastrointestinal Liner System, also called an “endoluminalliner,” duodenal mucosal resurfacing, also referred to as duodenalablation, partial bypass of the duodenum, involving duodeno-ileal orduodeno-jejunal anastomosis, vagal nerve blockade, and/or pyloroplasty).

A bariatric procedure (i.e., bariatric surgery) typically involves anyof the foregoing: partially or completely bypassing the duodenum and/ordecreasing nutrient exposure to the duodenum, increasing the rapidity ofnutrient transit to the lower part of the intestines (often specificallythe ileum), and/or otherwise increasing ileal nutrient exposure.Bariatric surgery may be intended for weight loss or metabolic benefit(such as resolution of diabetes), or both. Such weight loss or metabolicprocedures, referred to herein as “bariatric procedures” may enhancesecretion of GLP-1 from the distal small intestine, especially theileum, leading to elevated insulin secretion, and in some patientshypoglycemia. The patient may be referred to as a “post bariatricsurgery” patient or “post-RYGB.”

In another embodiment, the patient has previously had a relatedmetabolic procedure. As but one example, in one embodiment, the patienttreated has previously had a non-bariatric surgical procedure involvingthe gastrointestinal system (including but not limited to esophagectomy,for example for treatment of esophageal cancer, Nissen Fundoplication,for example for treatment of gastroesophageal reflux, or gastrectomy,for example for treatment of gastric cancer) and so may be referred toherein as “post gastrointestinal surgery.”

In another embodiment, the patient treated is prediabetic and/or insulinresistant and may benefit from prevention of pancreatic hyperstimulationfrom oral carbohydrate ingestion leading to post-prandial hypoglycemia.In another embodiment, a treated patient has a congenital, acquired, orinduced form of hyperinsulinemic hypoglycemia, such as congenitalhyperinsulinism or sometimes referred to as congenital nesidioblastosis.

In a preferred embodiment, however, the patient has had bariatricsurgery to aid in weight loss and/or metabolic control and has sufferedhypoglycemic excursions requiring urgent medical attention; suchpatients, as demonstrated conclusively in the examples below, canbenefit markedly from treatment with a subcutaneously administeredformulation of exendin(9-39) in accordance with the invention.

A typical adult patient with hyperinsulinemic hypoglycemia will presentwithin 10 years of bariatric and/or other gastrointestinal surgery withsymptoms of hypoglycemia (e.g. palpitations, tremor, weakness, sweating,confusion, fatigue, and/or blurred vision) within 5 hours of eating thatare associated with a plasma glucose of ≤60 mg/dL and immediateresolution with carbohydrate intake. Many patients experienceneuroglycopenic symptoms, such as altered mental status, loss ofconsciousness, or seizures. Hyperinsulinemia (>2 uU/mL or 13.9 pmol/L)may be documented in the proper laboratory setting at the time of thehypoglycemic event. However, documentation of hyperinsulinemia is notalways possible due to logistical challenges associated with thistesting (which involves induced hypoglycemia) and concerns over patientsafety.

Physicians skilled in the art will recognize from this disclosure thatthe methods of the invention provide effective treatment, such that aphysician following the same prescribing information herein can expecttherapeutic benefit will be achieved in patients whom, for treatment ofvarying underlying conditions, have had surgical manipulation of thegastrointestinal anatomy, and resultant secondary hyperinsulinemichypoglycemia.

Accordingly, to illustrate, the methods of the invention can be used totreat patients such as: 1) a patient whom, due to gastroesophagealreflux, underwent a Nissen Fundoplication procedure, and subsequentlydeveloped secondary hyperinsulinemic hypoglycemia; 2) a patient whom,due to a malignant gastric tumor (e.g. adenocarcinoma, gastrointestinalstromal tumor (GIST), or lymphoma), required partial or completegastrectomy, with or without any of the foregoing gastric reconstructiveprocedures: Bilroth I, Bilroth II, RYGB, or Jejunal interposition,developed secondary hyperinsulinemic hypoglycemia; and 3) a patientwhom, due to a tumor involving the esophagus or the esophageal gastricjunction (EGJ), underwent an esophagectomy, developed secondaryhyperinsulinemic hypoglycemia.

Those skilled in the art will further appreciate that patients withhypoglycemia due to endogenous, acquired, or congenital hyperinsulinism(“endogenous hyperinsulinemia” as used herein, refers to any suchcondition not caused by bariatric surgery or GI surgery) can benefitfrom application of the methods of the invention. Hypoglycemia in theseinstances can be severe, even life-threatening. Acquired hyperinsulinismmay result from insulinomas, autoimmune syndromes, reactivehypoglycemia, adult nesidioblastosis, or gastric dumping syndrome (notdue to bariatric or GI surgery). Congenital hyperinsulinism may manifestin the newborn period, or many years later. Accordingly, the methods andformulations of the invention include methods to treat such conditions.In the case of hyperinsulinemia resulting from an insulinoma andcongenital hyperinsulinism, a sustained release formulation and/or animmediate release formulation that is administered continuously, such asvia a subcutaneous pump, would be employed, with particular emphasis onthe prevention of nocturnal hyperinsulinemia.

In similar fashion, hyperinsulinism may further be induced as amedicinal side-effect of, for example, a GLP-1 agonist, such asexenatide, liraglutide, lixisenatide, albiglutide, and dulaglutide.Accordingly, the methods and formulations of the invention includemethods to treat overdoses with such drugs.

In some cases, patients with hyperinsulinemic hypoglycemia may alsopresent with cumulative hyperinsulinemic hypoglycemia-associatedcognitive impairment. Accordingly, the methods and formulations of theinvention include methods to treat or prevent a worsening of cognitiveimpairment in such patients. Further, in pediatric and adult patientsalike, acute and chronic hypoglycemia may be associated with morbiditiesnot only such as cognitive impairment, but also depression, heartpalpitations/tachycardia, and potentially other conditions, all of whichmay be reduced or prevented by preventing hypoglycemia by administrationof a GLP1A, such as exendin(9-39), as described herein forpost-bariatric patients suffering from hyperinsulinemia/hypoglycemia. Insome diabetic patients, severe hypoglycemia has repeatedly beenassociated with increased total and cardiovascular mortality risk. Thus,prevention of severe hypoglycemia is an important clinical goal for bothhospitalized and non-hospitalized patients, and the present inventionprovides methods and formulations useful for both groups of patients.

3.2 Treatment Parameters

In some embodiments, compositions comprising a therapeutically effectivedose of the GLP1A, exendin(9-39), are administered to a patient in needthereof for the treatment or prevention of hyperinsulinemichypoglycemia.

3.2.1. Administration Route

According to the invention, exendin(9-39) is administered bysubcutaneous administration (e.g., subcutaneous injection). Sites ofinjection, include, but are not limited to, injection in the thigh,abdomen, upper arm region, or upper buttock region.

3.2.2 Administration Dose

As discussed in Examples below and elsewhere herein, patients withhyperinsulinemic hypoglycemia may be treated by BID SC administration ofexendin(9-39) at therapeutically effective doses of 30 mg or lower(e.g., about 10 mg to 30 mg, 10 mg to 25 mg, 10 mg to 20 mg, 15 mg to 20mg, 10 mg to 17.5 mg, and 10 mg to 15 mg). Exemplary doses include 10mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg or 30 mg. Insome embodiments, the therapeutically effective amount of exendin(9-39)that is administered (e.g., in BID dosing) is selected from 10 mg, 15mg, 17.5 mg, and 20 mg. In some embodiments, a therapeutically effectivedose of exendin(9-39) or range of doses will vary depending upon theneeds and physical attributes of the patient. It will be understood by aperson of ordinary skill in the art that the doses described herein canbe administered at varying concentrations, including but not limited tothe injectate concentrations described in Section 3.2.4.1 below.

3.2.3 Administration Schedule

Advantageously, exendin(9-39) may be subcutaneously administered BID totreat hyperinsulinemic hypoglycemia. In some embodiments exendin(9-39)is subcutaneously administered QD.

3.2.3.1 BID Administration

BID (twice per day) administration is well known in the medical arts. Insome embodiments BID doses are administered (e.g., self-administered) atabout 12 hour intervals (e.g., 7 a.m. and 7 p.m.). However, shorter(e.g., 8 a.m. and 6 p.m.) or longer (e.g., 7 a.m. and 10 p.m.) intervalsbetween administrations are possible provided the administrations are atleast about 6 hours apart. Preferably the administrations are at leastabout 7 hours, 8 hours, 9 hours, 10 hours or 11 hours apart. Preferablythe administrations are not more than about 15 hours apart.

3.2.3.2 Timing of Administration and Relationship to Meals

In one aspect of the invention, an immediate-release formulation ofexendin(9-39) is provided as a subcutaneous injectable formulation thatis administered prior to the administration of a meal. For example, insome embodiments, exendin(9-39) is administered within 60-150 minutes(e.g., within 90-120 minutes) prior to morning and evening meals (orbefore the two main meals of the day, approximately 6 hours or moreapart). In some embodiments, exendin(9-39) is administered at least onehour prior to the morning meal. Using BID dosing 60-150 minutes (e.g.,within 90-120 minutes) prior to the morning and evening meals, the peakGLP-1 plasma concentration occurring approximately 30-60 minutespost-meal will be countered by sufficient exendin(9-39) plasmaconcentrations at that time to prevent GLP-1 induced hyperinsulinemia.

In another embodiment the BID dosing will be a morning and eveningadministration with a morning administration after wakening in themorning and evening administration about 12 hours later (in someembodiments, about 12-14 hours, about 12-16 hours later, or about 9-15hours later). The morning administration may be before or after themorning meal (breakfast). In this embodiment, the dosing schedule isindependent of (i.e., not based on, or dictated by) the timing of meals.In some embodiments the morning administration is within a specifiedtime before and/or after the morning meal (e.g. one hour before and/orone hour after breakfast). In some embodiments the morningadministration is before or after the morning meal, as discussed above,and the evening administration is prior to retiring for the night(bedtime) such as between the evening meal and bedtime, or within 1, 2,or 3 hours of bedtime.

In a related embodiment the dosing schedule is semi-independent ofmealtimes. For example, in the semi-independent schedule the morningdose is administered on a predetermined schedule relative to the morningmeal and the evening dose is scheduled at a time independent of the timeof the evening meal (e.g., about 12 hours after the morningadministration without regard to the time of the evening meal).

Without intending to be bound by a specific mechanism, it is believedthat the schedule, dose, route and formulations of the invention allowthe evening administration to provide additional protection atbreakfast, and the morning administration to provide protection duringthe day (e.g., lunch, dinner, or multiple small meals during the day).Advantageously, subcutaneous BID administration of a therapeuticallyeffective dose of exendin(9-39) is protective even when not timed tocoincide with meals. In contrast, as demonstrated in EXAMPLE 2, an IVbolus injection of 7,500 pmol/kg exendin(9-39) reversed hypoglycemiaonly if timed to coincide with the peak GLP-1 plasma concentrations. Thedosing approaches set forth herein provides considerably moreflexibility to the patient than alternative approaches, resulting inincreased compliance and a superior quality of life for the patient.

3.2.4 Administration Formulation 3.2.4.1 Injectate Concentration andVolume

Surprisingly, certain pharmacokinetic parameters of SC BID exendin(9-39)administration can be modified by selecting the concentration ofexendin(9-39) in the injectate. As described in the Examples,subcutaneous injection of a low concentration formulation results in ashorter Tmax (i.e., a faster rise to Cmax) relative to a higherconcentration. Subcutaneous injection of a high concentrationformulation results in a lower Cmax, a longer Tmax, and longer half-liferelative to a lower concentration. See FIG. 7A and Table 4.

For purposes of this invention a concentration less than 20 mg/ml is alow concentration, e.g., 4-20 mg/ml, preferably about 10-20 mg/ml, andoften about 8-16 mg/ml, most often about 13-16 mg/ml, and very often 15mg/ml. The low concentration formulation results in a pharmacokineticprofile useful for BID administration. As shown in FIG. 7B and Table 5(Example 3), subcutaneous administration of exendin(9-39) at varyingdoses but equivalent concentrations of about 13-16 mg/ml resulted in afavorable immediate release formulation with a Cmax greater than apreferred steady state plasma exendin(9-39) concentration of at least100 ng/ml as measured by liquid chromatograph-mass spectrometry, at 10,20 and 30 mg doses.

For purposes of this invention a concentration greater than about 20mg/ml (e.g., 20-45 mg/ml) is considered a “high” concentration. As shownin FIG. 7A and Table 4, subcutaneous injection of a relatively moreconcentrated solution, for example in a range inclusive of and exceeding20 mg/ml, e.g., 20-40 mg/ml, will result in a lower Cmax, with a longerhalf-life. For example, as shown in FIG. 7A and Table 4, subcutaneousadministration of exendin(9-39) at a dose of about 20 mg andconcentration of about 24 mg/ml exhibited a significantly longerhalf-life than subcutaneously administered exendin(9-39) at a dose ofabout 10 mg and concentration of about 16 mg/ml (9.14 hours vs. 3.60hours). In some embodiments, a more highly concentrated solution ofexendin(9-39) results in an exendin(9-39) plasma Cmax that is lower thana relatively lower concentration formulation but which is still greaterthan a preferred steady state plasma exendin(9-39) concentration of 70ng/ml or greater (e.g., as shown in FIG. 7A and Table 4 for the “10×”(approximately 20 mg) dose as compared to the “5×” (approximately 10 mg)dose). Thus, a more concentrated solution may be more amenable to lessfrequent dosing, e.g., QD dosing, or to BID dosing that is not tied tomeals.

In some embodiments, exendin(9-39) is subcutaneously administered at aconcentration of about 4-25 mg/ml, about 4-20 mg/ml, about 10-25 mg/ml,about 10-20 mg/ml, about 10-18 mg/ml, about 8-16 mg/ml, about 12-20mg/ml, about 10-15 mg/ml, or about 13-16 mg/ml (e.g., about 4 mg/ml,about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9mg/ml, about 10 mg/ml, about 11 mg/ml, about 12 mg/ml, about 13 mg/ml,about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 17 mg/ml, about 18mg/ml, about 19 mg/ml, about 20 mg/ml, about 21 mg/ml, about 22 mg/ml,about 23 mg/ml, about 24 mg/ml, or about 25 mg/ml).

In some embodiments, exendin(9-39) is subcutaneously administered at aconcentration in the range of about 13 mg/ml to about 16 mg/ml. In someembodiments, exendin(9-39) is subcutaneously administered at aconcentration of about 15 mg/ml.

As shown in FIG. 7B and Table 5, both a relatively lower dose of 10 mgand a relatively higher dose of 30 mg yielded a Cmax greater than thepreferred steady state plasma exendin(9-39) concentration of 70 ng/ml orgreater and were efficacious in reversing hyperinsulinemic hypoglycemiawhen administered at approximately equal concentrations in the range ofabout 13-16 mg/ml. Thus, in one aspect, a relatively lower dose ofexendin(9-39) (e.g., a dose of about 5-10 mg, e.g., about 5 mg, about7.5 mg, or about 10 mg) can be efficacious in treating hyperinsulinemichypoglycemia by adjusting the exendin(9-39) solution to an appropriateconcentration as described herein. In some embodiments, a relativelylower dose of exendin(9-39) (e.g., a dose of about 5-10 mg, e.g., about5 mg, about 7.5 mg, or about 10 mg) is administered at a concentrationof at least about 10 mg/ml, e.g., at a concentration in the range ofabout 13-16 mg/ml, e.g., at a concentration of about 15 mg/ml.

Generally exendin(9-39) is subcutaneously administered at aconcentration sufficient to result in a steady state plasmaexendin(9-39) concentration of at least 70 ng/ml, at least 100 ng/ml, orat least 150 ng/ml as measured by liquid chromatograph-massspectrometry. In some embodiments, exendin(9-39) is subcutaneouslyadministered at a concentration sufficient to result in a steady stateplasma exendin(9-39) concentration of about 100-200 ng/ml. In someembodiments, exendin(9-39) is subcutaneously administered at aconcentration sufficient to result in a steady state plasmaexendin(9-39) concentration of at least 70 ng/ml up to 250 ng/ml.

Table A below provides exemplary therapeutically effective exendin(9-39)formulations:

TABLE A Exendin(9-39) Concentration  1 10-30 mg 10-25 mg/mL  2 10-30 mg10-20 mg/mL  3 10-30 mg 10-18 mg/mL  4 10-30 mg 13-16 mg/mL  5 10-25 mg10-25 mg/mL  6 10-25 mg 10-20 mg/mL  7 10-25 mg 10-18 mg/mL  8 10-25 mg13-16 mg/mL  9 10-20 mg 10-25 mg/mL 10 10-20 mg 10-20 mg/mL 11 10-20 mg10-18 mg/mL 12 10-20 mg 13-16 mg/mL 13 10-15 mg 10-25 mg/mL 14 10-15 mg10-20 mg/mL 15 10-15 mg 10-18 mg/mL 16 10-15 mg 13-16 mg/mL 17 15-30 mg10-25 mg/mL 18 15-30 mg 10-20 mg/mL 19 15-30 mg 10-18 mg/mL 20 15-30 mg13-16 mg/mL 21 15-25 mg 10-25 mg/mL 22 15-25 mg 10-20 mg/mL 23 15-25 mg10-18 mg/mL 24 15-25 mg 13-16 mg/mL 25 15-20 mg 10-25 mg/mL 26 15-20 mg10-20 mg/mL 27 15-20 mg 10-18 mg/mL 28 15-20 mg 13-16 mg/mL

In some embodiments, each administration of a BID subcutaneousadministration of exendin(9-39) results in an exendin(9-39) Cmax of atleast 100 ng/ml. In some embodiments, for example for patients havingrelatively higher GLP-1 levels or having greater beta-cell sensitivityto GLP-1, each administration of a BID subcutaneous administration ofexendin(9-39) results in an exendin(9-39) Cmax of at least 150 ng/ml.

In some embodiments, the exendin(9-39) injectate comprises an exendin(9-39) dose and concentration that, when administered, results in steadystate plasma exendin(9-39) concentration of at least 70 ng/ml,preferably at least 100 ng/ml, or even more preferably at least 150ng/ml, as measured by LCMS. In some embodiments, the exendin(9-39)formulation has such a dose and concentration that results in steadystate plasma exendin(9-39) concentration of 100-250 ng/ml, e.g., 100-200ng/ml, 100-150 ng/ml, or 150-200 ng/ml.

In some embodiments, each dose is administered in a total volume rangingfrom 0.25-2 ml injectate, with most patients administering an injectionvolume ranging from 0.5-1.5 ml, e.g., 0.7-1 ml.

3.2.4.2 Injectate Formulation

Exendin(9-39) may be administered in any pharmaceutically acceptableform. In some embodiments, exendin(9-39) is formulated with apharmaceutically acceptable diluent or carrier that is suitable forsubcutaneous administration. Examples of pharmaceutically acceptablediluents or carriers include, but are not limited to, water, saline, andisotonic buffer solutions. In some embodiments the injectate formulationfurther comprises one or more additional excipients such aspreservatives and pH adjustment agents.

In one approach exendin(9-39) is formulated in normal saline (0.9%saline). In one approach the exendin(9-39) is formulated with anantimicrobial preservative, a tonicity-adjusting agent, such asmannitol, and/or a buffer (e.g., to bring the solution to a pH of about4-5).

As shown in Example 3 and FIG. 7A, administration of a lower dose (10mg, or “5×”) resulted in a higher exendin(9-39) Cmax than higher dosesformulated in the same volume of solution (i.e., having a higherconcentration). Without intending to be bound by a particular mechanism,this may be a result of aggregation (e.g., dimer or higher multimerformation). Thus, in one approach exendin(9-39) is formulated with anagent to reduce aggregation or dimer formation such as a surfactant(e.g., a non-ionic surfactant, such as a polysorbate or a poloxamer),polyol, or sugar, or by optimizing the pH and/or ionic strength of thesolution.

In one aspect, exendin(9-39) is formulated for immediate release. In oneapproach, exendin(9-39) is formulated as an injectable,immediate-release formulation of exendin(9-39) using a formulation thatis used to deliver exenatide, marketed as BYETTA™ (see U.S. Pat. Nos.5,424,286; 6,858,576; 6,872,700; 6,902,744; 6,956,026; 7,297,761;7,521,423; and 7,741,269, incorporated herein by reference).

In another aspect, exendin(9-39) is formulated for extended release,i.e., an extended release formulation, such that, when administered, theformulation ensures that the active drug product has a lasting presencein the blood throughout the targeted time period in the course oftreatment. Use of these formulations and methods allows plasma glucosehomeostasis to be maintained with fewer subcutaneous injections,relative to immediate release formulations. In some embodiments,exendin(9-39) is formulated with microspheres or nano-lipocapsules,which provide for sustained and extended release profiles. In someembodiments, exendin(9-39) is formulated with slowly erodingmicrospheres. Such microspheres include, for example and withoutlimitation, those made with a biopolymer, such as Poly(lactic-co-glycolic acid) (PLGA) or its equivalent. Such formulationsprovide for release of drug over an extended period of time (1-10weeks). To prepare the formulation, exendin(9-39) is loaded into themicrospheres, and the formulation provides that exendin is steadilyreleased over time as the matrix materials degrade. These microspherescan be formulated to minimize drug bursts and maintain a steady releaseprofile. In some embodiments, exendin(9-39) is encapsulated intonano-lipocapsules to prepare another formulation of the invention, whichprovides similar sustained and extended drug release. These formulationsare provided in a variety of particle and capsule sizes andcompositions, providing the physician a variety of rapid, medium, andslow release profile formulations to optimize therapy for individualpatients.

In one approach, exendin(9-39) is formulated as an injectable,extended-release formulation of exendin(9-39) using a formulation thatis used to deliver exenatide, marketed as BYDUREON™ (see U.S. Pat. Nos.5,424,286; 6,479,065; 6,495,164; 6,667,061; 6,824,822; 6,858,576;6,872,700; 6,956,026; 7,223,440; 7,456,254; 7,563,871; 7,612,176;7,741,269; 8,216,180; 8,329,648; 8,431,685; 8,439,864; 8,461,105; and8,906,851, incorporated herein by reference).

In another approach, exendin(9-39) is formulated using a formulationthat is used to deliver liraglutide, delivered in a daily dose, marketedas Victoza™ (see U.S. Pat. Nos. 6,004,297; 6,268,343; 6,458,924;7,235,627; 8,114,833; and 8,846,612).

Other formulations of the invention having a variety of differentfeatures and advantages may be made in accordance with the teachings ofU.S. Pat. Nos. 8,445,647; 8,895,033; 8,969,293; 8,299,025; and8,546,326; and U.S. Patent Application Publication Nos. 2015/0258016;2015/0238568; 2015/0057227; 2015/0056285; 2014/0309168; 2014/0256626;2013/0252894; 2013/0195939; and 2013/0172250, incorporated herein byreference, substituting exendin(9-39) for the active pharmaceuticalingredient described.

In some embodiments, exendin(9-39) is formulated as a sterile, preservedisotonic solution in a unit or multi-dose glass vial or ampule foradministration with the use of a syringe, similar to the glucagonemergency kit. In some embodiments, the exendin(9-39) is provided as aninjectable suspension in a single-dose tray containing a vial ofexendin(9-39), a vial connector, a prefilled diluent syringe, and one ormore needles.

In some embodiments, exendin(9-39) is formulated as a sterile, preservedisotonic solution in a glass cartridge pen-injector device. Suchcompositions, for example and without limitation contain 5-30 mg ofexendin(9-39), an appropriate volume of an antimicrobial preservative, atonicity-adjusting agent, such as mannitol, and a buffer to bring thesolution to a pH of about 4-5.

In some instances, the formulation of exendin(9-39) is provided as aninjectable suspension in a single-dose pen containing exendin(9-39), adiluent, and one or more needles. In some instances, microneedles coatedwith or containing the formulation of exendin(9-39) are used.

In some embodiments, exendin(9-39) is formulated as a sterile, preservedisotonic solution in a glass cartridge pen-injector device. Suchcompositions, for example and without limitation contain 5-30 mg ofexendin(9-39), an appropriate volume of an antimicrobial preservative, atonicity-adjusting agent, such as mannitol, and a buffer to bring thesolution to a pH of about 4-5.

3.2.5 Different Evening and Morning Injectates

In some embodiments, twice-per-day administration comprisesadministering a morning injectate and an evening injectate that containdifferent exendin(9-39) doses and/or different concentrations ofexendin(9-39). Generally, each of the injectates has an exendin(9-39)amount and concentration within the ranges described herein. However, inthis embodiment, the amount of exendin(9-39) in the eveningadministration is greater than the amount in the morning injectateand/or the exendin(9-39) concentration in the evening injectate isgreater than the concentration of exendin(9-39) in the morninginjectate. In some embodiments the two injectates will have differentquantities, the same concentration, of. In some embodiments the twoinjectates will have the same amount of exendin(9-39) but differentconcentration. In some embodiments both the concentration and amount ofexendin(9-39) will be different.

Without intending to be bound by a particular mechanism, the increasedamount of exendin(9-39) administered in the evening may provide higherexendin(9-39) levels at the time of the morning meal. Without intendingto be bound by a particular mechanism, the increased concentration ofexendin(9-39) is expected to result in a more “flat” plasmaconcentration profile, including a longer time to Tmax, for a moresustained effect at the time of the morning meal.

In some embodiments the amount of exendin(9-39) in the evening injectateis 5 mg to 10 mg greater than the amount in the morning injectate. Insome embodiments the amount of exendin(9-39) in the evening injectate is5 mg greater than the amount in the morning injectate. In someembodiments the amount of exendin(9-39) in the evening injectate is 10mg greater than the amount in the morning injectate. In some embodimentsthe amount of exendin(9-39) in the morning injectate is 10 mg, 15 mg, or20 mg.

In some embodiments the concentration of exendin(9-39) in the eveninginjectate is 5 mg/ml-10 mg/ml greater than the amount in the morninginjectate. In some embodiments the concentration of exendin(9-39) in theevening injectate is about 5 mg/ml greater than the amount in themorning injectate. In some embodiments the concentration ofexendin(9-39) in the evening injectate is about 10 mg/ml greater thanthe amount in the morning injectate. In some embodiments theconcentration of exendin(9-39) in the morning injectate is 10-16 mg/mland the concentration of exendin(9-39) in the evening injectate ishigher and is in the range of 15-20 mg. In some cases, the amount ofexendin(9-39) in the evening injectate is 5 mg-10 mg greater than theamount in the morning injectate. In some such cases the amount ofexendin(9-39) in the morning injectate is 10 mg and the amount in theevening injectate is 15 mg. In some such cases the amount ofexendin(9-39) in the morning injectate is 10 mg and the amount in theevening injectate is 20 mg. In some such cases the amount ofexendin(9-39) in the morning injectate is 15 mg and the amount in theevening injectate is 20 mg. In some such cases the amount ofexendin(9-39) in the morning injectate is 15 mg and the amount in theevening injectate is 25 mg. In some such cases the amount ofexendin(9-39) in the morning injectate is 20 mg and the amount in theevening injectate is 25 mg. In some such cases the amount ofexendin(9-39) in the morning injectate is 20 mg and the amount in theevening injectate is 30 mg.

In some cases, the concentration of exendin(9-39) in the morninginjectate is not the same as the concentration of exendin(9-39) in theevening injectate. For example, in one approach the concentration ofexendin(9-39) in morning injectate is 15 mg/ml and the concentration ofexendin(9-39) in the evening injectate is 20 mg/ml.

In some cases the exendin(9-39) amount and concentration of the morningand evening injectates are selected such that the exendin(9-39) Tmaxafter the evening administration is longer than the Tmax after themorning administration.

In one approach, the evening injectate is prepared or formulated tofavor multimerization (e.g., dimerization) or precipitation of theexendin(9-39). Administration of the injectate at bedtime can delayabsorption, producing a slower release profile compared to the morningadministration, resulting in an advantageous basal morning level of atleast 30 ng/mL Methods for preparing compositions comprisingmultimerized proteins are known. For example, the addition of a basicprotein, such as protamine, to the exendin(9-39) preparation can favorformation of multimer peptide configurations. Alternatively,multimerization can be achieved by precipitating the exendin(9-39) outof solution, for example through the addition of salts, such as zincsalts, such that the molar ratio of the salt with respect toexendin(9-39) is greater than 1, so as to reduce the solubility ofexendin(9-39) in a neutral solvent. In this approach, raising the pH(for example to 7.4), in the presence of such salts, can be used tofavor precipitation of the peptide. Thus, in some embodiments the levelof aggregation or multimerization in the evening injectate is greaterthan the level in the morning injectate. In some embodiments theexendin(9-39) is in a less soluble form in the evening injectatecompared to the morning injectate.

3.2.6 Duration of Therapy

Patients may receive therapy for a predetermined time, an indefinitetime, or until an endpoint is reached. Treatment may be continued on acontinuous daily or weekly basis for at least two to three months, sixmonths, one year, or longer. In some embodiments, therapy is for atleast 30 days, at least 60 days, at least 90 days, at least 120 days, atleast 150 days, or at least 180 days. In some embodiments, treatment iscontinued for at least 6 months, at least 7 months, at least 8 months,at least 9 months, at least 10 months, at least 11 months, or at leastone year. In some embodiments, treatment is continued for the rest ofthe patient's life or until administration is no longer effective inmaintaining normal plasma glucose levels to provide meaningfultherapeutic benefit. In some embodiments, adult patients (60-100 kg ormore) will receive therapeutic benefit from a single dose ofexendin(9-39).

3.3 Delivery Devices

Devices such as injectable pen devices and pumps suitable forsubcutaneous injections are well known. Such devices may be used todeliver the exendin(9-39) formulations described hereinabove accordingto the methods described herein.

3.3.1 Injectable Pen Device

In some embodiments, exendin(9-39) is administered using an injectablepen device that may be pre-programmed to deliver a fixed dosage amount.In some embodiments, the device is pre-programmed to deliver a fixeddosage ranging from 5-30 mg, e.g., 10-20 mg, or 7.5-15 mg, dependingupon the needs and physical attributes of the patient. In someembodiments, the exendin(9-39) is formulated as an immediate releasepreparation, and is packaged, for example, in the form of a single ordual-chamber pen device (e.g., a 1 to 5 mL dual chamber pen—either adisposable pen or one that reloads disposable cartridges).

The drug product can be supplied as a freeze-dried lyophilized powder,stored in a 1 to 3 mL or larger, e.g., 5 mL, dual-chamber cartridge thatis compatible with a disposable pen injector (see, for example, theYpsomed dual chamber cartridge/pen injector: www.ypsomed.com). Dosestrengths can be conveniently made available to patients, including forexample doses in the range of 5-30 mg of exendin(9-39), to bereconstituted in a volume of 0.25-2.0 ml normal saline per dose, orother pharmaceutically acceptable diluent suitable for subcutaneousadministration.

In some embodiments, the drug product is supplied as individualinjectable pen devices that are pre-programmed to deliver a fixed dosageamount, in which the morning dosage amount and the evening dosage amountare different amounts and/or concentrations. For example, in someembodiments, a first pen (e.g., for morning administration) delivers adose in the range of 5-15 mg (e.g., a dose of 5 mg, 7.5 mg, 10 mg, 12.5,or 15 mg) and a second pen (e.g., for evening administration) delivers ahigher dose in the range of 15-20 mg (e.g., a dose of 15 mg, 17.5 mg, or20 mg). In some embodiments, the first pen delivers a dose of 10 mg andthe second pen delivers a dose of 15 mg. In some embodiments a first pendelivers a dose of 15 mg and the second pen delivers a dose of 20 mg.

In another aspect, the present invention provides kits comprisingindividual injectable pen devices as described herein. In someembodiments, a kit comprises a plurality of individual injectable pendevices (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pens in a kit). Insome embodiments, the kit comprises two or more individual injectablepen devices that are pre-programmed to deliver a fixed dosage amount, inwhich the morning dosage amount and the evening dosage amount aredifferent amounts and/or concentrations. For example, in someembodiments, the kit comprises a first pen (e.g., for morningadministration) that delivers a dose in the range of 5-15 mg (e.g., adose of 5 mg, 7.5 mg, 10 mg, 12.5, or 15 mg) and further comprises asecond pen (e.g., for evening administration) that delivers a higherdose in the range of 15-20 mg (e.g., a dose of 15 mg, 17.5 mg, or 20mg). In some embodiments, the kit comprises a first pen that delivers adose of 10 mg and a second pen that delivers a dose of 15 mg. In someembodiments, the kit comprises a first pen that delivers a dose of 15 mgand a second pen that delivers a dose of 20 mg.

3.3.2 Subcutaneous Pump

In one embodiment, the methods of the invention comprise the use of asubcutaneous pump, and the invention provides such pumps containingexendin(9-39) formulated as described herein for subcutaneous delivery.This methodology is generally very convenient for the patient.Compositions for such methods provided by the invention include solutionformulations and freeze dried lyophilized powder for reconstitution.See, e.g., Kumareswaran et al., Discovery Medicine, 2012, 13:159-170,incorporated by reference herein.

3.4 Treatment Outcomes

In some embodiments, patients treated with the compositions and methodsdescribed herein exhibit an improvement in one or more symptoms ofhypoglycemia, including but not limited to neuroglycopenic symptoms,beta-adrenergic symptoms, or plasma glucose levels.

In some embodiments, treatment in the typical adult or pediatric patientrefers to treatment such that the postprandial plasma glucose nadir ismaintained above a concentration of approximately 55 mg/dl (3.0mmol/liter) based upon the Endocrine Society's Clinical Guidelines(Journal of Clinical Endocrinology & Metabolism, 2009, 94(3):709-728),and symptoms of hypoglycemia are reduced. Ideally, normal plasma glucoseconcentrations are maintained, with those skilled in the art recognizingthat in humans a blood glucose level of 65 mg/dl or greater ispreferred.

In some embodiments, treatment in a patient refers to treatment suchthat at least a 15% increase in postprandial plasma glucose nadir isachieved relative to baseline (e.g., before the onset of treatment). Insome embodiments, treatment in a patient refers to treatment such thatfor a patient having a postprandial plasma glucose nadir≤50 mg/dl atbaseline (e.g., before the onset of treatment), an increase inpostprandial plasma glucose nadir to ≥55 mg/dl is achieved relative tobaseline.

Plasma glucose nadir can be measured, for example, by oral glucosetolerance test (OGTT) or meal tolerance test (MTT) as described herein.In some embodiments, treatment in a patient refers to treatment suchthat a statistically significant decrease in the severity of one or moresymptoms of hypoglycemia overall during a OGTT or MTT and/or ofneuroglycopenic symptoms elicited during the glucose “fall” period ofOGTT or MTT is achieved relative to baseline (e.g., before the onset oftreatment).

3.5 Dosage Escalation

Some physicians may desire to treat with a low or initiating (starting)dose (e.g., 5-7.5 mg), escalate to an increased if the initiating dosedoes not result in acceptable glycemic control, and maintain theinitiating dose if glycemic control is sufficient.

In some embodiments, a starting dose of 10 mg exendin(9-39) in a morningdose and 10 mg exendin(9-39) in an evening dose is administered to thesubject. If this dose does not result in sufficient coverage in themorning (e.g., does not result in sufficient glycemic control at thetime of the morning meal), the evening dose may be increased, e.g., to15 mg exendin(9-39) as the evening dose. In some embodiments, a startingdose of 15 mg exendin(9-39) in a morning dose and 15 mg exendin(9-39) inan evening dose is administered to the subject. If this dose does notresult in sufficient coverage in the morning, the evening dose may beincreased, e.g., to 20 mg exendin(9-39) as the evening dose.

These and other benefits of the invention will be appreciated in greaterdepth upon contemplation of the examples below and accompanying figures,which demonstrate that administration of exendin(9-39) as describedherein can provide immediate and significant benefit to post-bariatricpatients suffering hypoglycemic excursions after consuming normalamounts of glucose. Based upon American Society of Metabolic andBariatric Surgery (ASMBS) recommendations (seeasmbs.org/patients/life-after-bariatric-surgery), post-bariatric surgerypatients are encouraged to limit their carbohydrate intake to 50 gramsper day or less. Patients in the examples provided were administered 75grams of carbohydrate within a 20 minute period of time, amounting to1.5-fold the total ASMBS recommended daily intake. Thus, based upon thesuccess demonstrated in the examples wherein administration ofexendin(9-39) prevented hypoglycemia and markedly improved symptomsafter a high carbohydrate load, under ordinary conditions, similar orgreater efficacy would be anticipated.

4. Examples 4.1 Example 1: Continuous IV Infusion of Exendin(9-39)Effectively Reverses Hyperinsulinemic Hypoglycemia and AssociatedSymptoms

A randomized placebo-controlled blinded cross-over Phase 1 study wasconducted to determine whether continuous IV infusion of exendin(9-39)can effectively reverse hyperinsulinemic hypoglycemia and associatedsymptoms. Exendin(9-39) was acquired as a lyophilized peptide:exendin(9-39) acetate 10 mg/vial from Bachem (Clinalfa, Laufelfingen,Switzerland). For preparation of the IV infusate, lyophilizedexendin(9-39) was solubilized with 20 ml 0.9% normal saline (NS) forevery 10 mg peptide, then diluted in 100 ml 0.9% NS and 50 ml of 25%human serum albumin, in a PVC-free, DEHP-free 1L infusion bag. The bagwas covered with an opaque IV bag cover to aid with blinding. Anidentical-appearing bag was prepared, constituting the placebo infusate,containing the same volume of infusate (NS only) without the presence ofpeptide or albumin. Eight patients with hyperinsulinemic hypoglycemiawere randomized to receive an infusion of placebo and an infusion ofexendin(9-39) in cross-over design during an oral glucose tolerance test(OGTT) on two separate days separated by no greater than 2 weeks.Patients were asked to fast for 12 hours prior to the infusion of studydrug or placebo, and infusions and OGTTs were carried out the Center forTranslational Research Unit (CTRU) at Stanford University. On the day ofadmission to CTRU, 2 IV lines were placed for infusion of study drug andblood collection. Fasting blood was drawn at T-40 minutes. At T-30minutes, an IV bolus of 7,500 pmol/kg exendin(9-39) or placebo wasadministered over 1 minute, while a continuous IV infusion ofexendin(9-39) at a rate of 500 pmol/kg/min (providing an infusion doseof about 0.35 mg/kg) or placebo (0.9% saline) was initiated and run for210 minutes. At T+0 minutes an OGTT was initiated, wherein patients wereinstructed to consume a 75 g glucola drink over 20 minutes.

Plasma samples were collected at T-40, T+0, T+30, T+45, T+60, T+90,T+105, T+120, T+150, T+180 and at each timepoint immediately taken tothe laboratory for processing. The following assays were then conducted:glucose, insulin, GLP-1, GIP, glucagon, and exendin(9-39). If glucoselevels dropped to 50 mg/dL or less, the test was stopped andinvestigators intervened as needed to normalize glucose. At T-40 andconcomitant with timed blood draws, a graded symptom questionnaire wascompleted repetitively by patients. This questionnaire was adapted fromtwo validated hypoglycemia assessment tools, by segregating symptomsinto three clear factors: autonomic, neuroglycopenic, and malaise, andthen by adding a severity gradation scale, such that patients rated theseverity of each reported symptoms from 1-5 (1: least severe; 5: mostsevere).

As shown in FIG. 1B and Table 1, patients exhibited an average glucosenadir of approximately 80 mg/dL during exendin(9-39) infusion, ascompared to a nadir of <50 mg/dL during placebo infusion. Patients alsoexhibited a marked decrease in plasma insulin concentrations duringexendin(9-39) infusion (see FIG. 1A and Table 1).

Metabolic responses, including plasma GLP-1, GIP, and glucagonresponses, were measured as shown in FIG. 2 A-C, and Table 2. Althougharea under the curve (AUC) values were calculated as shown in Tables 1and 2, the presentation of the data graphically, as presented in FIGS. 1and 2 , is more informative because subject OGTTs were stoppedprematurely if they became hypoglycemic (as they did in 100% of casesduring placebo infusion). For calculation of AUC in cases of prematurecessation of the OGTT, the last value was carried forward. Patients werealso assessed for hypoglycemic symptoms during exendin(9-39) infusionvs. placebo infusion. As shown in FIG. 3 , continuous exendin(9-39)infusion substantially improved symptoms of hypoglycemia, asdemonstrated by the dramatically reduced total hypoglycemic symptomassessment score. Additionally, to isolate symptoms associated withglucose rise and fall, two subscores were included: the “Glucose Fall”score, which encompasses symptoms associated with the fall in glucose tonadir, and the “Glucose Rise” score, which encompasses symptomsassociated with the rise in glucose to peak.

The results demonstrate that continuous IV infusion of exendin(9-39)effectively reverses hyperinsulinemic hypoglycemia and associatedsymptoms.

4.2 Example 2: Single IV Bolus Injection of Exendin(9-39) ReversesHypoglycemia Only if Timed Coincide with Peak GLP-1 PlasmaConcentrations

A trial was performed to assess whether a single bolus dose ofexendin(9-39) was able to prevent hypoglycemia in a 75 gram OGTT withsubjects with hyperinsulinemic hypoglycemia. Two subjects withhyperinsulinemic hypoglycemia were admitted to the research clinic aftera 12 hour overnight fast. An IV bolus of 7,500 pmol/kg exendin(9-39) wasprepared as in Example 1. The subjects consumed a 75 gram glucola atT=0. GLP-1 levels are predicted to peak about 60 min after theadministration of glucola (see, Myint et al., European Journal ofEndocrinology, 2012, 166:951-955; see also FIG. 4D). After consuming theglucola, the subjects were infused intravenously with an IV bolus ofexendin(9-39) over 1 minute, with the timing of the IV bolusadministration relative to the 75 gram OGTT altered on different days,as follows: T=0, T+20, and T+50. Plasma was assayed at T-40, T+0, T+30,T+45, T+60, T+90, T+105, T+120, T+150, T+180 and at each timepointimmediately taken to the laboratory for processing. Measurements weretaken for glucose, insulin, GLP-1, GIP, glucagon, and exendin(9-39).Bioavailability/PK profile of IV exendin(9-39) was evaluated by Cmax,Tmax, AUC0-∞, AUClast, VZ, CL, and T_(1/2). Exendin(9-39) concentrationwas measured by radioimmunoassay (MA) as described in Kielgast et al.,Diabetes, 2011, 60:1599-1607.

As shown in FIGS. 4A-C, dosing of the IV bolus of exendin(9-39) at 0minutes or 20 minutes following administration of glucola did notprevent hypoglycemia, whereas dosing at 50 minutes after administrationof glucola did prevent hypoglycemia. See, figure legend. FIGS. 4A-Ddemonstrates that peak plasma exendin(9-39) concentrations in the rangeof 500-600 nMol/L by radioimmunoassay at the time of peak plasma GLP-1concentrations are required to avoid a glucose nadir below 50 mg/dL. Theresults shown in FIG. 4A-D suggest that in the absence of continuous IVinfusion, or in the absence of an IV bolus timed precisely to the peakpredicted GLP-1 plasma concentrations, hypoglycemia cannot be averted.

Exendin(9-39) plasma levels can be measured using a radioimmunoassay(MA) generally as described in Kielgast et al., Diabetes, 2011,60:159-1607. Exendin(9-39) plasma levels can be measured using liquidchromatography-mass spectrometry (LCMS) methodology generally asdescribed in Lasaosa et al., J. Chromatogr B Analyt Technol Biomed LifeSci, 2014, 0:186-191. We refer to both methods in the discussion herein,and both methods are used in the scientific literature. We observed thatmeasurement of plasma exendin(9-39) values using RIA were significantlyhigher than values determined using LCMS. We believe the LCMS values aremore accurate. For definitional purposes, a claimed exendin(9-39)concentration (e.g., Cmax) refers to the absolute quantity ofExendin(9-39) which may be determined by LCMS or another equallyquantitative method.

FIG. 5A depicts an average of eight patients' plasma exendin(9-39)concentrations at various timepoints following a 7,500 pmol/kg IV bolusof exendin(9-39) at T-30 minutes, followed by continuous IV fusion at arate of 500 pmol/kg/min over 210 minutes as described in Example 1. Seegraph line with error bars. It has also been reported that in healthysubjects an intravenous infusion of exendin(9-39) at 500 pmol/kg/minfully reverses the glucose lowering effect of GLP-1. See, Edwards etal., Diabetes, 1999, 48:86-93. Based on the measured plasmaexendin(9-39) concentrations as shown in FIG. 5A, a steady plasmaexendin(9-39) concentration of approximately 500 nmol/L (as measured byradioimmunoassay) or of approximately 140 nmol/L (as measured by LCMS)is presumed to be required for efficacy.

FIG. 5A also shows the projected exendin(9-39) plasma concentration thatwould be expected from administering a single IV bolus of 7,500 pmol/kgexendin(9-39) at T-30 minutes. As previously reported, the half-life ofa single dose of intravenously administered exendin(9-39) is about 33.5minutes (see, Edwards et al., Diabetes, 1999, 48:86-93). Extrapolatingfrom the exendin(9-39) concentration measured at T=0 (about 300 nmol/Las measured by RIA) it was concluded that the exendin concentration atT=−30 is about 600 nmol/L given the half-life of intravenouslyadministered exendin(9-39). In view of the projected pharmacokineticresponse for exendin(9-39) and the time course of the development ofhypoglycemia following a meal (typically 1-3 hours after meals, withpeak GLP-1 levels expected at about 60 minutes after the meal), a singleIV bolus dose administered prior to or with a meal would likely not beeffective for treatment of hyperinsulinemic hypoglycemia, because theexendin(9-39) plasma concentration would be expected to be very low atthe predicted time of peak GLP-1 levels. Furthermore, even if an IVbolus having a higher dose of exendin(9-39) were administered, it wouldbe expected to exhibit similar pharmacokinetic properties of a shorthalf-life and rapid elimination from plasma. In view of the time coursefor the development of hypoglycemia and the lag between the time of ameal and the projected peak GLP-1 levels, even an IV bolus having ahigher dose of exendin(9-39) would not be expected to be efficacious inaverting hypoglycemia unless precisely timed with predicted peak plasmaGLP-1 levels.

4.3 Example 3: A Single Dose of Subcutaneously Injected Exendin(9-39)Effectively Reverses Hyperinsulinemic Hypoglycemia and AssociatedSymptoms

As described above in Example 1, it was found that an IV bolus of 7,500pmol/kg exendin(9-39) plus a continuous IV infusion of exendin(9-39) ata rate of 500 pmol/kg/min over 210 minutes was efficacious in reversinghyperinsulinemic hypoglycemia and associated symptoms. For the peptideexenatide, it has been reported that the absorption kinetics ofexenatide in rats most closely approximates human absorption kinetics.See, Chen et al., Interspecies Modeling Pharm Res., 2013, 30:751-760.Rat intravenous and subcutaneous dose escalation pharmacokinetic datapredicts that in humans, the Cmax of subcutaneously administeredexendin(9-39) would be substantially lower than the Cmax ofintravenously administered exendin(9-39). Accordingly, it was expectedthat a higher dose of exendin(9-39) would be needed for subcutaneousadministration, as compared to intravenous administration, in order forexendin(9-39), to be effective in reversing hyperinsulinemichypoglycemia.

To compare the pharmacokinetic parameters of intravenously orsubcutaneously administered exendin(9-39), a single IV bolus of 7,500pmol/kg exendin(9-39) or a single subcutaneous injection of 7,500pmol/kg exendin(9-39) was administered in one subject on two separatedays. The IV bolus consisted of 0.025 mg/kg of lyophilized exendin(9-39)(which equates to a dose of approximately 2 mg for an 80 kg patient)solubilized in 20 ml per 10 mg exendin(9-39) (approximately 4 ml normalsaline) and then diluted in 100 ml 0.9% normal saline for every 10 mgexendin(9-39) (approximately 20 ml 0.9% normal saline), to whichapproximately 10 ml 25% human serum albumin was added (50 ml 25% humanserum albumin for every 10 mg exendin(9-39)), for a total IV bolusinfusion volume of approximately 34 ml. The IV bolus infusion wasadministered over 1 minute. The subcutaneous injection consisted of0.025 mg/kg of lyophilized exendin(9-39) (which equates to a dose ofapproximately 2 mg for an 80 kg patient) solubilized in 0.2 ml normalsaline and further diluted in 0.5 ml normal saline to a total volume of0.7 ml for subcutaneous injection in the arm. Plasma exendin(9-39)concentrations were measured by liquid chromatography-mass spectrometry(LCMS) as described in Lasaosa et al., supra Example 1. As shown in FIG.5B, the Cmax that was observed in subcutaneous administration ofexendin(9-39) was significantly lower than the Cmax observed inintravenous administration of exendin(9-39), further supporting thehypothesis that for subcutaneous administration, a higher dose ofexendin(9-39) would be required for preventing hyperinsulinemichypoglycemia, as compared to efficacious doses of intravenouslyadministered exendin(9-39).

A single ascending dose (SAD) study was performed to assess thepharmacokinetics, efficacy, and local tolerability of administeringexendin(9-39) by subcutaneous injection. For the SAD study, ninesubjects with hyperinsulinemic hypoglycemia were randomized to one offour experiments, each representing one of four subcutaneous doses ofexendin(9-39): 7,500 pmol/kg, 37,500 pmol/kg, 75,000 pmol/kg, or 112,500pmol/kg. Lyophilized exendin(9-39) acetate 10 mg/vial from Bachem(Clinalfa, Laufelfingen, Switzerland) was acquired for each experiment,with each 10 mg vial solubilized in 200 μl normal saline, then furtherdiluted with normal saline to a total dose of 7,500 pmol/kg, 37,500pmol/kg, 75,000 pmol/kg, or 112,500 pmol/kg (2.0 mg, 10 mg, 20 mg, or 30mg of exendin(9-39), respectively, based on a patient weight of 80 kg).The total volume of each injectate was held constant, with furtherdilution of injectate as required to result in a total volume ofinjectate of 0.7 ml. Of the nine subjects, five subjects were randomizedto receive one subcutaneous injection of 7,500 pmol/kg, 37,500 pmol/kg,75,000 pmol/kg, or 112,500 pmol/kg (2, 10, 20, and 30 mg, respectively,based on an 80 kg patient) in a volume of 0.7 ml normal saline, and foursubjects received two or more 0.7 ml injections of 75,000 pmol/kg, or112,500 pmol/kg in order to maintain an injectate concentration of about15 mg/ml or less.

Subjects fasted overnight for 12 hours and were admitted to the researchclinic. One IV line was placed in the patient for blood collection.Fasting blood was drawn. Subjects were injected subcutaneously in theabdomen with the dose of exendin(9-39) to which they were randomized,and were blinded as to which dose they were receiving. For the subjectsreceiving a dose of 37,500 pmol/kg, 75,000 pmol/kg, or 112,500 pmol/kg,an OGTT was initiated at T+0 minutes, wherein patients were instructedto consume a 75 g glucola drink over 20 minutes. Plasma samples werecollected at T-10, T-0, T+15, T+30, T+45, T+60, T+75, T+90, T+105,T+120, T+135, T+150, T+165, T+180, T+210, T+240, T+300, T+480, andT+1440, and at each timepoint the samples were immediately taken to thelaboratory for processing.

The following parameters were evaluated: 1) plasma glucose, insulin,glucagon, GLP-1, and GIP concentration; 2) bioavailability/PK profile ofsubcutaneous exendin(9-39): Cmax, Tmax, AUC0-∞, AUClast, VZ, CL,T_(1/2), and bioavailability; 3) local tolerability after subcutaneousinjection of exendin(9-39) utilizing a Visual Analog Scale (VAS) and aNumeric Rating Scale (NRS); and 4) local swelling as measured by caliperat timed intervals along the long and short axes of the swelling andbump height. Patients were also assessed for hypoglycemic symptoms usinga graded symptom questionnaire in which patients rated the severity ofspecifically recited hypoglycemia symptoms from 1-5 (1: least severe; 5:most severe) at specific timepoints, from which a “Glucose Rise” score,“Glucose Fall” score, and “All Timepoints” score were calculated.Exendin(9-39) concentration was measured by liquid chromatograph-massspectrometry as described by Lasaosa et al., supra Example 1.

For the 8 subjects who were administered a subcutaneous dose of 37,500pmol/kg, 75,000 pmol/kg, or 112,500 pmol/kg, the plasma glucoseconcentrations were measured during the OGTT. (The dose of 7,500 pmol/kgthat was administered to subject 1 was presumed to be subtherapeutic,and so an OGTT was not administered to this subject.) For the remainingeight subjects who were administered a subcutaneous dose ofexendin(9-39) and an OGTT, none of the subjects became hypoglycemicafter subcutaneous injection at doses ranging from 35,000-112,500pmol/kg. Thus, prevention of hypoglycemia was achieved at allsubcutaneous dose levels. In contrast, all of the subjects receivingplacebo became hypoglycemic during the OGTT. As shown in FIG. 6 , theaverage plasma nadir for the 8 subjects administered a subcutaneous doseof exendin(9-39) was 78 mg/dL, versus <50 mg/dL for the placebo.Additionally, as shown in Table 3, the average subject symptomaticresponse was significantly improved for the subjects who wereadministered the subcutaneous dose of exendin(9-39) and OGTT, asmeasured by a dramatically reduced Overall Symptom Score (14.6 vs. 20.6for placebo) and Symptom Fall Score (4 vs. 22 for placebo).

As shown in Table 4, subcutaneous administration of exendin(9-39) as asingle injection at a dose ranging from 37,500 pmol/kg to 112,500pmol/kg (approximately 10-30 mg) and a constant volume of 0.7 ml wasefficacious for preventing hypoglycemia, for example as shown by theplasma glucose nadir. An injectate concentration of approximately 15mg/ml (the 37,500 pmol/kg dose) resulted in the greatest pharmacodynamicresponse, as defined by Cmax and dose-normalized Cmax. For the subjectswho were subcutaneously administered exendin(9-39) at relativelyequivalent concentrations (approximately 13-16 mg/ml), as shown in Table5, exendin(9-39) administration was efficacious in preventinghypoglycemia, for example as shown by the plasma glucose nadir. Forthese patients, Table 5 shows that there was an increasingly favorablePK response with increasing dose, as defined by Cmax and T_(1/2).

As shown in FIG. 8 , a strong correlation was found between the percentincrease in plasma glucose nadir concentrations (comparing plasmaglucose nadir after subcutaneous injection of exendin(9-39) to baselineplasma glucose nadir) and the peak plasma exendin(9-39) concentrations(Cmax).

Surprisingly, the clinical efficacy achieved for the subcutaneouslyadministered doses of exendin(9-39) tested in this SAD study wasequivalent to the efficacy that was achieved by continuous IV infusionof larger quantities of exendin(9-39) as described in Example 1, asshown for example in Table 3 for the plasma glucose nadir, AUC glucose,and the Symptom Fall Score parameters.

In view of the efficacy of the subcutaneously administered dose levelsas demonstrated in this example, the efficacy, safety, tolerability, andpharmacokinetics of subcutaneous administration of exendin(9-39) over adefined time period is evaluated, for example, as described in Example 4and Example 5 below.

4.4 Example 4: Multi-Ascending Dose Trial to Assess the Efficacy,Tolerability, and Pharmacokinetic Profile of BID Exendin(9-39) inPatients with Post-Bariatric Hyperinsulinemic Hypoglycemia

This example describes a Phase 2a clinical study protocol for evaluatingthe safety, tolerability, efficacy, and pharmacokinetic profile of BIDexendin(9-39) administered subcutaneously over 3 days to patients withpost-bariatric hyperinsulinemic hypoglycemia.

TABLE B Study objectives and endpoints: Objective Endpoint Primary: Toevaluate the treatment effect on Response rate in plasma glucose nadir,defined as plasma glucose of SC BID Ex9 proportion of patients in eachdose arm with no plasma glucose ≤50 mg/dL at any timepoint from 0-180minutes during OGTT on Day 3 of treatment vs. on Day 0. Secondary: Toevaluate the treatment effect on Improvement in composite symptom scoreas symptoms of hypoglycemia of SC compared to baseline during OGTT onDay 3 of BID Ex9 treatment vs. on Day 0. To assess the pharmacokineticsof Plasma PK parameters include AUC_(0-12h), C_(max), SC BID Ex9 at eachdose level T_(max), T_(1/2), and C_(trough), after SC injection. Toassess the safety and tolerability AEs, laboratory parameters, vitalsigns; NMR of SC BID Ex9 at each dose level score, VAS score.

Overview:

The study is a single-blinded, dose-randomized, cross-over design studythat is being conducted at the Stanford University School of Medicine.All subject visits will take place in the Clinical and TranslationalResearch Unit (CTRU). Sixteen to twenty eligible subjects will beassigned to one of five dose levels (2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg)to receive subcutaneous injection of BID exendin(9-39) administered forthree days. After a baseline Oral Glucose Tolerance Test (OGTT) isconducted on Day 0 wherein metabolic and symptomatic analyses willoccur, subjects will return to the research clinic on Day 1 to initiatea BID dosing schedule for 3 days. During this time, subjects will returndaily for fasting labs in the morning, a morning dose, PK sampling, andan evening trough sample, followed immediately by the second daily doseat T+720 min. Safety, tolerability, and pharmacokinetic parameters willbe measured on a daily basis for the full three day duration of thestudy, after which a repeat OGTT is performed on the morning of Day 3after the morning dose to evaluate for efficacy (no hypoglycemia andreduction in composite symptom score). Day 4 will consist solely ofclinical safety monitoring with a plasma trough drawn 1440 minutes afterthe last Day 3 injection. This study if properly conducted is expectedto demonstrate that BID dosing can result in meaningful therapeuticactivity in each dosing arm. See, FIG. 9 .

Randomization/Blinding:

For the first four subjects dosed, the subjects were randomly assignedto one of the following dose levels: 2.5 mg, 5 mg, or 10 mg. Theremaining subjects will be randomly assigned to one of the followingdose levels: 10 mg, 15 mg, or 20 mg. All subjects will remain blindedthroughout. With the exception of the PI and sub-investigator who willremain un-blinded for safety purposes, all site personnel includingnurses and study coordinators, who conduct patient symptom surveys, willremain masked to treatment assignment.

Study Drug Preparation and Dispensation:

All doses will be prepared to a total concentration of ≤15 mg/ml ofexendin(9-39) in normal saline. Each 10 mg vial of lyophilizedexendin(9-39) will be diluted in either 1 ml normal saline if a 10 mg/mlconcentration is administered, or 0.7 ml normal saline if a 14 mg/mlconcentration is administered. For doses requiring total volume ofinjection>1 ml, 2 injections will be employed.

Oral Glucose Tolerance Test (OGTT):

The OGTT will consist of administration of one 75 mg gram glucola drinkwith 1 gram of crushed acetaminophen to be consumed over 20 minutes.

Assays:

Metabolic: glucose, c-peptide, insulin, GLP-1, GIP, glucagon; PK:AUC₀₋₇₂₀, C_(max), T_(max), T_(1/2), C_(trough).

Anticipated PK Profile:

It was anticipated, based on the prior results for a single subcutaneousinjection (as shown in Example 3), that after administration of a 5 mg,10 mg, 15 mg, or 20 mg dose the plasma concentration of exendin(9-39)would return to <20 ng/mL or even close to 0 ng/mL within 720 minutes ofinjection. However, based on the intermediate results of BID dosing for3 days as shown in FIG. 10 and as discussed below, it is expected thatadministration of a 10-30 mg dose will result in a higher nadir, such asa nadir of about 30-80 ng/ml within 720 minutes after injection.

It is expected that a dosage of 5 mg BID, 10 mg, 15 mg or 20 mg BIDexendin(9-39) will demonstrate a therapeutic benefit for one or morepatients in the 3-day trial. A “therapeutic benefit” may be defined withreference to effect on plasma glucose. For example, in some instances adosage of exendin(9-39) provides a therapeutic benefit for a patientwhen the patient has no plasma glucose≤50 mg/dL at any timepoint from0-180 minutes during OGTT on Day 3 of treatment as compared to Day 0. Insome instances a dosage of exendin(9-39) provides a therapeutic benefitfor a patient when the patient has at least a 15% increase in plasmaglucose nadir during OGTT on Day 3 relative to Day 0. In some instancesa dosage of exendin(9-39) provides a therapeutic benefit for a patientwhen the patient has at least a 15% increase in AUC glucose. In someinstances a dosage of exendin(9-39) provides a therapeutic benefit for apatient when the patient has a statistically significant decrease in theseverity of one or more symptoms of hypoglycemia overall during the OGTTand/or of neuroglycopenic symptoms elicited during the glucose “Fall”period of the OGTT relative to Day 0. In some instances a dosage ofexendin(9-39) provides a therapeutic benefit for a patient having aplasma glucose nadir≤50 mg/dL at baseline when the patient exhibits aplasma glucose nadir≥55 mg/dL after a defined treatment period (e.g.,after a 3 day treatment period).

Intermediate Results

Four subjects were randomized to one of three dose levels (2.5 mg, 5 mg,or 10 mg) to receive subcutaneous injection of BID exendin(9-39)administered for three days.

Patient 1 was administered a dose of 5 mg at a concentration of 10 mg in1 ml, subcutaneously administered in the abdomen. For Patient 1, a 13.1%increase in AUC glucose was observed as compared to baseline, buthypoglycemia was not prevented, as defined by plasma glucose≤50 mg/dL.Patient 2 was administered a dose of 2.5 mg at a concentration of 10 mgin 1 ml, subcutaneously administered in the abdomen. For Patient 2, an8.8% increase in AUC glucose was observed, but hypoglycemia was notprevented, as defined by plasma glucose≤50 mg/dL. Patient 3 wasadministered a dose of 5 mg at a concentration of 10 mg in 1 ml,subcutaneously administered in the arm. For Patient 3, a 16.3% increasein AUC glucose was observed, but hypoglycemia was not prevented, asdefined by plasma glucose≤50 mg/dL.

Patient 4 was administered a dose of 10 mg at a concentration of 10 mgin 1 ml, subcutaneously administered in the arm. For this patient,hypoglycemia was not prevented, as defined by plasma glucose≤50 mg/dL.

These intermediate pharmacodynamic results demonstrate an increasingtherapeutic benefit, as defined by % increase in glucose AUC withincreasing doses administered, with one of the two patients dosed with 5mg experiencing a greater than 15% increase in AUC glucose as comparedto AUC glucose during a baseline oral glucose tolerance test. Whilehypoglycemia as defined by plasma glucose≤50 mg/dL was not prevented, atherapeutic dose response was achieved, illustrating that doses of 10-30mg will result in improved glycemic control, as further shown by Example3 and FIG. 8 .

The pharmacokinetic parameters obtained from the 3-day BID dosing ofPatients 1-4 are shown in Table 6 below. The single subject dosed at 10mg for 3 days was severely disabled, experiencing daily episodes ofsymptomatic neuroglycopenia, and requiring placement of gastrostomy tubeinto the remnant stomach for route of nutrient administration. Theseintermediate results demonstrate that total exendin(9-39) exposureincreases with increasing dose. As shown in Table 6, AUC was increasedby about 1.5-1.7-fold, and Cmax was increased by about 50% with a 5 mgdose escalation from 5 mg to 10 mg. Similar degrees of increase areexpected to be observed for AUC and Cmax with an escalation to a 15 mgdose. For a 15 mg dose, a Cmax value is expected to be in thetherapeutically effective range of approximately 150-200 ng/ml. Interimpharmacokinetic results from this 3-day trial also demonstrate that onaverage, AUC plasma concentrations increase with increasing days of BIDdosing. A higher trough was observed at Day 3 than at Day 1, suggestingseveral days (e.g., 3-5 days) may be required to reach steady state.Thus, the results of this study support efficacy of the 15 mg dose atDay 3 of treatment. The results of this study also support efficacy ofthe 10 mg dose in less severely disabled patients and/or with longer(e.g., 5 days) treatment.

Comparison of the pharmacokinetic responses to abdominal versus arminjection of 5 mg doses in patient 1 and patient 3 demonstrates that aquicker absorption profile with higher exposure as defined by Cmax (seeTable 6) can be achieved by administration of the injectate into an areawith less subcutaneous fat, as may be the abdominal subcutaneous areaafter bariatric surgery weight loss. A slower absorption profile withlonger exposure, as defined by AUClast (see Table 6) can be achieved byadministration of the same dose into an area with relatively moresubcutaneous fat, such as the arm area may have after bariatric surgeryweight loss.

4.5 Example 5: Multiple Doses of Subcutaneously Injected Exendin(9-39)Safely and Effectively Reverse Hyperinsulinemic Hypoglycemia

This example demonstrates the method of the invention in which amulti-site multi-ascending dose (MAD) format is used to evaluate theefficacy, safety, and pharmacokinetics of a 28-day trial of immediaterelease subcutaneous exendin(9-39) administered BID in patients withsevere post-bariatric hypoglycemia. The primary objective of this trialis to demonstrate the efficacy of exendin(9-39) on plasma glucose levelsduring a 3-hour oral Glucose Tolerance Test (OGTT) at the end of 4-weektreatment. This trial is also intended to demonstrate the efficacy ofexendin(9-39) on the frequency and severity of hypoglycemia incidenceand associated symptoms in patients with severe post-bariatrichypoglycemia. This trial also demonstrates the pharmacokinetics andpharmacodynamics of exendin(9-39) at each dose level. Furthermore, thistrial demonstrates the safety and tolerability profile of the immediaterelease subcutaneous formulation of exendin(9-39) in patients withsevere post-bariatric hypoglycemia.

This is a multi-center, double-blind, randomized, placebo-controlled,parallel-group, two exendin(9-39) dose levels, phase 2 study in patientswith severe post-bariatric hypoglycemia. Approximately 36 patients willbe recruited. Eligible patients will have a confirmed diagnosis ofsevere hypoglycemia post-bariatric via Whipple's triad and OGTT. Thestudy is divided into three phases, as follows:

Screening Phase:

All potential subjects will complete an oral glucose tolerance test(OGTT), wherein if plasma glucose falls to less than or equal to 60mg/dL and all other eligibility criteria are met, the patient will beallowed to enroll in the study. In cases of out of range laboratoryvalues, with the exception of laboratory tests related to re-feedingsyndrome, subjects are permitted to re-screen one time.

4-Week Randomized Treatment (RT) Period:

All enrolled subjects will participate in a 4-week randomized treatmentperiod wherein subjects will be randomized to one of two exendin(9-39)doses (e.g., 10 mg and 20 mg, 10 mg and 15 mg, or 15 mg and 20 mg)administered BID or matching placebo of the 2 doses. The ratio oftreatment assignment to the first exendin(9-39) BID dose, the secondexendin(9-39) BID dose, the first matching placebo dose, and the secondmatching placebo dose will be 2:2:1:1. During the RT period, thesubjects will undergo continuous glucose monitoring wearing Dexcoms athome.

Open-Label Extension (OLE) Period:

All patients completing Week 4 of the randomized treatment period andexperiencing benefit with exendin(9-39) at the end of RT will beeligible to enter the OLE period. During the OLE period, the doseadministered will either be an optimal fixed dose level selected at theend of the randomized treatment period of the study or up-titrated to 20mg BID until any of the following occur: completed 12 months of theopen-label extension; unacceptable toxicity; lack of efficacy; protocoldeviation; patient withdrew consent; lost to follow-up; death; and studydiscontinues per the sponsor.

The primary efficacy endpoint is measured as the response rate in plasmaglucose level at the end of the 4-week RT, defined as the proportion ofpatients either (1) without plasma glucose≤55 mg/dL for patients whoseglucose nadir is ≤50 mg/dL at baseline OGTT; or (2) without plasmaglucose≤60 mg/dL for patients whose glucose nadir is 55-≤60 mg/dL atbaseline OGTT. Secondary efficacy endpoints are measured as theimprovement in neuroglycopenic symptom score during OGTT at the end ofRT (Week 4), where neuroglycopenic symptoms include inability toconcentrate, confusion, weakness, drowsiness, dizziness, blurred vision,difficulty speaking (modified from the Edinburgh Hypoglycemia Score,Hepburn 1991); the proportion of patients with severe hypoglycemiaduring the 4-week RT, where severe hypoglycemia is defined an eventrequiring assistance of another person to actively administercarbohydrates, glucagon, or take other corrective actions with a bloodglucose concentration of <50 mg/dL by continuous glucose monitoring(CGM); the proportion of patients with any hypoglycemia event betweenWeek 2 and Week 4, where hypoglycemia is defined as a plasma glucoseconcentration of ≤55 mg/dL by continuous glucose monitoring (CGM)[Hypoglycemia after Roux-En-Y gastric bypass: detection rates ofcontinuous glucose monitoring (CGM) versus mixed meal test Kefurt 2014];and the Change in Quality of life at Week 4 from baseline as evaluatedusing Short-Form 36 (SF-36) domain scores.

The pharmacokinetic and pharmacodynamics endpoints to be measuredinclude C_(max), T_(max), T_(1/2), C_(trough), AUC of exendin(9-39).Exploratory endpoints will include insulin (AUC, Peak, ISR, ICR),GLP-1/GIP, and glucagon concentrations.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

TABLE 1 Subject metabolic responses to OGTT with and without intravenousinfusion of Exendin (9 39) vs. non-surgical controls HH Placebo^(a) HHEx(9-39)^(a) Non-Surg controls^(a) (n = 8) (n = 8) P-Value^(b)P-value^(c) P-value^(d) Fasting plasma glucose (mg*dl⁻¹) 91.8 ± 1.2 94.7± 3.9 100.6 ± 4.3  0.414 0.06 0.322 Peak postprandial glucose (mg*dl⁻¹)235.4 ± 11.0 225.5 ± 15.1 152.3 ± 6.1  0.432 0.000 0.001 Time to peakglucose (min) 56.3 ± 3.8 37.5 ± 4.9 45.0 ± 5.7 0.011 0.120 0.334 Nadirpostprandial glucose (mg*dl⁻¹) 46.1 ± 1.9 78.7 ± 5.4 74.9 ± 3.8 0.0000.000 0.570 Time to hypoglycemia (min) 137.5 ± 5.3  NA NA NA NA NA Deltapeak-nadir glucose (mg*dl⁻¹) 189.3 ± 10.1 146.8 ± 15.3 77.4 ± 7.6 0.0120.000 0.001 Rate of glucose decline (mg*dl⁻¹*min⁻¹)  3.1 ± 0.5  1.1 ±0.3  0.5 ± 0.2 0.001 0.001 0.189 AUC glucose (0, 60)(mg*dl⁻¹*min⁻¹)10402.5 ± 309.3  10905.9 ± 624.1  8120.6 ± 287.1 0.278 0.000 0.001 AUCglucose(60, 180)(mg*dl⁻¹*min⁻¹) 11318.9 ± 573.3  15397.5 ± 1180.713346.3 ± 504.9  0.010 0.019 0.378 AUC glucose(0, 180)(mg*dl⁻¹*min⁻¹)21721.4 ± 701.8  26303.4 ± 1785.8 21466.9 ± 642.2  0.020 0.793 0.023Fasting plasma insulin (uU*ml⁻¹)  4.0 ± 19.2  3.2 ± 0.8 15.0 ± 1.2 0.2600.000 0.000 Peak postprandial insulin (uU*ml⁻¹) 200.3 ± 28.5  88.3 ±23.0 86.0 ± 8.3 0.000 0.002 0.928 Time to peak insulin (min) 60.0 ± 5.748.8 ± 5.5  67.5 ± 12.4 0.285 0.590 0.187 AUC insulin (0,60)(uU*ml⁻¹*min⁻¹) 6220.3 ± 766.4 3368.9 ± 832.4 3420.0 ± 375.7 0.0010.005 0.956 AUC insulin (60, 180)(uU*ml−1*min−1) 4591.6 ± 876.3 2462.2 ±524.8 6532.5 ± 607.9 0.038 0.090 0.000 AUC insulin (0,180)(uU*ml⁻¹*min⁻¹) 13605.5 ± 1819.0  5831.1 ± 1281.0 9952.5 ± 869.90.001 0.092 0.019 Insulin at glucose <55 mg/dl (uU*ml⁻¹) 17.5 ± 4.7 NANA NA NA NA Insulinogenic Index (0, 30)  1.2 ± 0.2  0.7 ± 0.2  1.2 ± 0.40.001 0.927 0.286 Insulinogenic Index (0, 60)  1.4 ± 0.2  0.6 ± 0.1  1.9± 0.4 0.000 0.239 0.006 ^(a)Data are presented as mean ± SEM^(b-d)P⁻value by two-sided student's t-test ^(b)P⁻value HH Placebo vs.HH Ex(9-39) ^(c)P⁻value HH Placebo vs. Non-Surg Controls ^(d)P⁻value HHEx(9-39) vs. Non-Surg Controls

TABLE 2 Subject incretin responses to OGTT with and without intravenousinfusion of Exendin (9-39) HH Placebo^(a) HH Ex(9-39)^(a) (n = 8)P-value^(b) Fasting GLP-1 (pmol*L⁻¹)  9.0 ± 0.4  9.4 ± 1.2 0.191 PeakGLP-1 (pmol*L⁻¹) 86.0 ± 6.1  82.3 ± 13.9 0.857 Time to peak GLP-1 (min)42.9 ± 6.1 38.6 ± 5.5 0.604 AUC GLP-1 (0, 60)(pmol*L ⁻¹*min⁻¹) 3270.0 ±214.1 3267.9 ± 606.8 0.998 AUC GLP-1 (60, 180)(pmol*L⁻¹*min⁻¹) 3135.7 ±265.7 3621.4 ± 416.0 0.429 AUC GLP-1 (0, 180)(pmol*L⁻¹*min⁻¹) 6405.7 ±423.0 6889.3 ± 941.2 0.700 Peak Insulin to Peak GLP-1 ratio  2.6 ± 0.1 1.3 ± 0.4 0.059 Fasting GIP (pmol*L−1*min−1) 14.4 ± 1.4 14.0 ± 1.50.824 Peak GIP (pmol*L−1*min−1)  93.1 ± 12.8  86.6 ± 12.0 0.005 Time topeak GIP (min) 42.9 ± 6.0 34.3 ± 4.3 0.356 AUC GIP (0,60)(pmol*L⁻¹*min⁻¹) 3831.4 ± 495.9 3267.9 ± 606.8 0.127 AUC GIP (60,180)(pmol*L⁻¹*min⁻¹) 3722.1 ± 168.9 3165.0 ± 396.9 0.084 AUC GIP (0,180)(pmol*L⁻¹*min⁻¹) 7553.6 ± 617.9 6420.0 ± 941.2 0.003 Peak Insulin toPeak GIP ratio 2.4 0.4 1.3 0.4 0.000 Fasting glucagon (pmol*L⁻¹) 40.5 ±2.8 41.9 ± 2.9 0.567 Peak glucagon (pmol*L⁻¹) 82.8 ± 6.7 92.3 ± 6.40.079 Time to peak glucagon (min)  60.0 ± 12.7  45.0 ± 11.3 0.470 AUCglucagon (0, 60)(pmol*L⁻¹*min⁻¹) 3796.6 ± 306.2 4430.5 ± 285.1 0.033 AUCglucagon (60, 180)(pmol*L⁻¹*min⁻¹) 7999.8 ± 912.2 8017.5 ± 696.3 0.981AUC glucagon (0, 180)(pmol*L⁻¹*min⁻¹) 11584.4 ± 1252.9 12019.3 ± 941.9 0.981

TABLE 3 Placebo^(a) SC Ex(9-39)^(a) IV Ex(9-39)^(a) (n = 8) (n = 8) (n =8) P-value^(b) P-value^(c) Subject metabolic response Fasting plasmaglucose (mg*dl⁻¹) 91.6 ± 1.7 94.5 ± 1.7 94.7 ± 3.9 0.125 0.966 Peakpostprandial glucose (mg*dl⁻¹) 229.3 ± 13.2 252.3 ± 23.7 225.5 ± 15.10.258 0.358 Time to peak glucose (min) 54.5 ± 3.7 52.5 ± 4.9 37.5 ± 4.90.351 0.049 Nadir postprandial glucose (mg*dl⁻¹) 47.7 ± 1.6 77.9 ± 4.178.7 ± 5.4 <.001 0.906 Time to hypoglycemia (min) 135.5 ± 4.9  NA NA NANA Delta peak-nadir glucose (mg*dl⁻¹) 182.0 ± 11.0 174.4 ± 24.2 146.8 ±15.3 0.588 0.351 Rate of glucose decline (mg*dl⁻¹*min⁻¹)  2.9 ± 0.4  2.4± 0.7  1.1 ± 0.3 0.402 0.117 AUC glucose (0, 60)(mg*dr⁻¹*min⁻¹) 10171.4± 334.6  11135.6 ± 704.4  10905.9 ± 624.1  0.140 0.811 AUC glucose(0,180)(mg*dr⁻¹*min⁻¹)  21106 ± 1001.6 27471.6 ± 1963.0 26303.4 ± 1785.80.007 0.667 Subject symptomatic response Overall SymptomScore{circumflex over ( )}  26 ± 3.3 14.6 ± 4.4  4.5 ± 2.2 0.006 0.057Symptom Rise Score+  11 ± 2.4 12.3 ± 3.7  4.3 ± 2.2 0.794 0.087 SymptomFall Score*  22 ± 3.5  4.0 ± 1.5  1.1 ± 0.4 0.001 0.091 ^(a)Data arepresented as mean ± SEM ^(b-c)P⁻value by two-sided paired student'st-test ^(b)P⁻value SC Ex(9-39) vs. Placebo ^(c)P⁻value SC Ex(9-39) vs.IV Ex(9-39)

TABLE 4 37500 pmol/kg 75000 pmol/kg 125000 pmol/kg n = 1 n = 2 n = 1Injectate Characteristics Fold-increase in dose relative to 7,500pmol/kg 5× 10× 15× Concentration (mg/ml) 15.714 23.57 41.43 Total doseadministered (mg) 11

 17 29 Volume per injection (ml) 0.7 0.7 0.7 Number of injections 1 1 1Subject Pharmacodynamic Response Fasting plasma glucose (mg * dl⁻¹) 9495.8 103.5 Peak postprandial glucose (mg * dl⁻¹) 244 226.8 311.0 Time topeak glucose (min) 60 60 60 Nadir postprandial glucose (mg * dl⁻¹) 8871.3 58.0 Delta peak-nadir glucose (mg * dl⁻¹) 156 155.5 253.0 Rate ofglucose decline (mg * dl⁻¹ * min⁻¹) 1 2.2 6.7 AUC glucose (0,60)(mg *dl⁻¹ * min⁻¹) 10815 9694 12713 AUC glucose(0,180)(mg * dl⁻¹ * min⁻¹)28482 25151 25718 Subject Pharmacokinetic Response Cmax (ng/ml) 114

 73.35 56 DN Cmax (ng/mL/mg) 10.36 4.45 1.93 Tmax (h) 3.25 6.25 5.00AUCINF [h * ng/ml] 1097

 1210 728 DN_AUC_(INF) [h * ng/ml/mg] 69

 63 24 AUC_(last) (h * hg/ml] 1084

 696 720 T^(1/2)(h) 3.60

 9.14 3.64 MRT_(last) [h]) 6.16

 5.74 6.62 MRT_(INF) [h] 6.45

 14.66 6.87

TABLE 5 37500 pmol/kg 75000 pmol/kg 112500 pmol/kg n = 1 n = 1 n = 2Injectate Characteristics Fold-increase in dose relative to 7,500pmol/kg Concentration (mg/ml) 15.714 14.71 13.48 Total dose administered(mg) 11 21

 29 Volume per injection (ml) 0.7 0.7 0.7 Number of injections 1 2 3Subject Pharmacodynamic Response Fasting plasma glucose (mg * dl⁻¹) 9490

 90.0 Peak postprandial glucose (mg * dl⁻¹) 244 187.0

 228.0 Time to peak glucose (min) 60 30

 45 Nadir postprandial glucose (mg * dl⁻¹) 88 75.5

 85.0 Delta peak-nadir glucose (mg * dl⁻¹) 156 111.5

 143.0 Rate of glucose decline (mg * dl⁻¹ * min⁻¹) 1 0.6

 1.5 AUC glucose (0,60)(mg * dl⁻¹ * min⁻¹) 10815 9540

 11063 AUC glucose(0,180)(mg * dl⁻¹ * min⁻¹) 28482 21023

 27274 Subject Pharmacokinetic Response Cmax (ng/ml) 114 158 229 DN Cmax(ng/mL/mg) 10.36 7.52 7.9 Tmax (h) 3.25 4.50 4.50 AUCINF [h * ng/ml]1097 1516 1885 DN_AUC_(INF) [h*ng/ml/mg] 69 101 67 AUC_(last) (h *ng/ml] 1084 900 1055 T^(1/2)(h) 3.60 4.59 4.87 MRT_(last) [h]) 6.16 4.394.69 MRT_(INF) [h] 6.45 8.55 9.24

TABLE 6 Dose 2.5 mg 5 mg 5 mg 10 mg Number of subjects dosed n = 1 n = 1n = 1 n = 1 Injectate Characteristics Dose (mg) 2.5 5 5 10 Concentration(mg/ml)   10 mg/ml  10 mg/ml  10 mg/ml 10 mg/ml Volume per injection(ml) 0.25 ml 0.5 ml 0.5 ml  1 ml Number of injections 1 1 1 1 Locationof administration abdomen abdomen arm arm Subject PharmacodynamicResponse % increase AUC glucose (treatment-baseline) 8.8 13.1 16.3 N/APlasma glucose nadir ≤50 mg/dL (Yes/No) Yes Yes Yes Yes SubjectPharmacokinetic Response Day 3 Cmax (ng/ml) 53.4 77.2 65 128 DN Cmax(ng/mL/mg) 21.36 15.44 13 12.8 Tmax Day 3 (h) 4 5 5 5 AUClast (h *ng/ml] 379.2 409.1 684.3 969 T1/2(h) 4.3 4.4 4.6 6.5

The invention claimed is:
 1. A method for treating hyperinsulinemichypoglycemia in a subject, the method comprising subcutaneouslyadministering to the subject in need thereof a therapeutically effectiveamount of exendin(9-39); wherein the therapeutically effective amount is10-30 mg of exendin(9-39); wherein the exendin(9-39) is administered ata concentration of 4-20 mg/ml; and wherein the subject exhibits one ormore of Whipple's triad, the occurrence of capillary glucose≤50 mg/dL atleast once per month, and a plasma glucose concentration of ≤60 mg/dLduring a tolerance test.
 2. The method of claim 1, wherein the tolerancetest is an oral glucose tolerance test or a meal tolerance test.
 3. Themethod of claim 1, wherein the subject exhibits a plasma glucoseconcentration of ≤55 mg/dL and plasma insulin≥3 uU/mL during thetolerance test, or wherein the subject exhibits a plasma glucoseconcentration of ≤55 mg/dL and c-peptide>0.3 mg/dL during the tolerancetest.
 4. The method of claim 1, wherein the therapeutically effectiveamount is 10-20 mg of exendin(9-39).
 5. The method of claim 1, whereinthe exendin(9-39) is administered once per day.
 6. The method of claim1, wherein the exendin(9-39) is administered twice per day.
 7. Themethod of claim 1, wherein the patient has previously had bariatricsurgery.
 8. The method of claim 7, wherein the bariatric surgery isRoux-en-Y gastric bypass, vertical sleeve gastrectomy, placement of anendosleeve device, duodenal mucosal resurfacing, partial bypass of theduodenum, vagal nerve blockade, or pyloroplasty.
 9. The method of claim1, wherein the patient has previously had gastrointestinal surgery. 10.The method of claim 9, wherein the gastrointestinal surgery isgastrectomy, Nissen Fundoplication, or esophagectomy.
 11. A method fortreating hyperinsulinemic hypoglycemia in a subject, the methodcomprising subcutaneously administering to the subject in need thereof atherapeutically effective amount of exendin(9-39), wherein thetherapeutically effective amount is 10-30 mg of exendin(9-39); whereinthe exendin(9-39) is administered at a concentration of 4-20 mg/ml; andwherein the subject is prediabetic and/or insulin resistant.
 12. Themethod of claim 11, wherein the subject has a congenital, acquired, orinduced form of hyperinsulinemic hypoglycemia.
 13. The method of claim11, wherein the therapeutically effective amount is 10-20 mg ofexendin(9-39).
 14. The method of claim 11, wherein the exendin(9-39) isadministered once per day.
 15. The method of claim 11, wherein theexendin(9-39) is administered twice per day.
 16. The method of claim 11,wherein the patient has previously had bariatric surgery.
 17. The methodof claim 16, wherein the bariatric surgery is Roux-en-Y gastric bypass,vertical sleeve gastrectomy, placement of an endosleeve device, duodenalmucosal resurfacing, partial bypass of the duodenum, vagal nerveblockade, or pyloroplasty.
 18. The method of claim 11, wherein thepatient has previously had gastrointestinal surgery.
 19. The method ofclaim 18, wherein the gastrointestinal surgery is gastrectomy, NissenFundoplication, or esophagectomy.
 20. A method for treatinghyperinsulinemic hypoglycemia in a subject, the method comprisingadministering to the subject in need thereof a therapeutically effectiveamount of exendin(9-39) twice-per-day (BID); wherein the therapeuticallyeffective amount is 10-30 mg of exendin(9-39); wherein the exendin(9-39)is administered at a concentration of 4-20 mg/ml; and wherein the seconddaily dose is administered 9 to 15 hours after the first daily dose. 21.The method of claim 20, wherein the therapeutically effective amount is10-20 mg of exendin(9-39).
 22. The method of claim 20, wherein thepatient has previously had bariatric surgery.
 23. The method of claim22, wherein the bariatric surgery is Roux-en-Y gastric bypass, verticalsleeve gastrectomy, placement of an endosleeve device, duodenal mucosalresurfacing, partial bypass of the duodenum, vagal nerve blockade, orpyloroplasty.
 24. The method of claim 20, wherein the patient haspreviously had gastrointestinal surgery.
 25. The method of claim 24,wherein the gastrointestinal surgery is gastrectomy, NissenFundoplication, or esophagectomy.